Recombinant sugarcane cystatin CaneCPI-5 promotes osteogenic differentiation.

Cysteine proteases orchestrate bone remodeling, and are inhibited by cystatins. In reinforcing our hypothesis that exogenous and naturally obtained inhibitors of cysteine proteases (cystatins) act on bone remodeling, we decided to challenge osteoblasts with sugarcane-derived cystatin (CaneCPI-5) for up to 7 days. To this end, we investigated molecular issues related to the decisive, preliminary stages of osteoblast biology, such as adhesion, migration, proliferation, and differentiation. Our data showed that CaneCPI-5 negatively modulates both cofilin phosphorylation at Ser03, and the increase in cytoskeleton remodeling during the adhesion mechanism, possibly as a prerequisite to controlling cell proliferation and migration. This is mainly because CaneCPI-5 also caused the overexpression of the CDK2 gene, and greater migration of osteoblasts. Extracellular matrix remodeling was also evaluated in this study by investigating matrix metalloproteinase (MMP) activities. Our data showed that CaneCPI-5 overstimulates both MMP-2 and MMP-9 activities, and suggested that this cellular event could be related to osteoblast differentiation. Additionally, differentiation mechanisms were better evaluated by investigating Osterix and alkaline phosphatase (ALP) genes, and bone morphogenetic protein (BMP) signaling members. Altogether, our data showed that CaneCPI-5 can trigger biological mechanisms related to osteoblast differentiation, and broaden the perspectives for better exploring biotechnological approaches for bone disorders.

c-Src kinase contributes on endothelial cells mechanotransduction in a heat shock protein 70-dependent turnover manner.

Shear stress changes are associated with a repertory of signaling cascade modulating vascular phenotype. As shear stress-related tensional forces might be associated with pathophysiological susceptibility, a more comprehensive molecular map needs to be addressed. Thus, we subjected human umbilical vein endothelial cells (HUVECs) to a circuit of different tensional forces in vitro considering the following three groups: (a) physiological blood flow shear stress condition (named Normo), (b) a hypertensive blood flow shear stress (named Hyper), and (c) these hyper-stressed cells were returned to Normo condition (named Return). The samples were properly collected to allow different methodologies analysis. Our data showed a pivotal involvement of c-Src on driving the mechanotransduction cascade by modulating signaling related with adhesion, survival (PI3K/Akt) and proliferative phenotype. Moreover, c-Src seems to develop important role during extracellular matrix remodeling. Additionally, proteomic analysis showed strong involvement of heat shock protein 70 (HSP70) in the hypertensive-stressed cells; it being significantly decreased in return phenotype. This result prompted us to investigate 20S proteasome as an intracellular proteolytic alternative route to promote the turnover of those proteins. Surprisingly, our data reveled significant overexpression of sets of proteasome subunit α-type (PSMA) and ß-type (PSMB) genes. In conjunction, our data showed c-Src as a pivotal protein to drive mechanotransduction in endothelial cells in a HSP70-dependent turnover scenario. Because shear patterns is associated with pathophysiological changes, such as atherosclerosis and hypertension, these results paved new road to understand the molecular mechanism on driving mechanotransduction in endothelial cells and, if drugable, these targets must be considered within pharmacological treatment optimization.

Improvement of lipase obtaining system by orange waste-based solid-state fermentation: production, characterization and application.

Lipases are an economic important group of biocatalysts that can be produced by some fungal under solid-state fermentation. Orange wastes are source of lipases and potential substrates for lipases production. This work assessed 19 fugal strains cultivated in Citrus sinensis cv. Hamlin orange wastes (peel, frit and core) for production of lipases in order to generate compounds with antioxidant, antimicrobial and cytotoxic properties. Fifteen of those fungi grew and produced lipases, mainly the Aspergillus brasiliensis [National Institute of Quality Control (INCQS) 40036]/frit system, which showed 99.58 U/g total lipase. The substrate with the highest production of lipase was frit with 26.67 and 78.91 U/g of total lipases produced on average by the 15 microorganisms. Aspergillus niger 01/frit (33.53 U/g) and Aspergillus niger (INCQS 40015)/frit (34.76 U/g) systems showed the highest specificity values in all the herein tested synthetic substrates with 4, 12 and 16 carbons. Analysis of the fatty acid profile of hydrolysis products obtained in the most prominent systems applied to corn and sunflower oils showed: palmitic acid, linoleic acid, oleic acid, and stearic acid. These acids showed antioxidant capacity of up to 58% DPPH (2,2-diphenyl-1-pierylhydrazyl) radical reduction and antibacterial activity against Escherichia coli, Listeria monocytogenes, Pseudomonas aureginosa, Salmonella Enteritidis and Staphylococcus aureus, as well as cytotoxicity to SCC9 cells (squamous cancer cells).

Bioremediation of cooking oil waste using lipases from wastes.

Cooking oil waste leads to well-known environmental impacts and its bioremediation by lipase-based enzymatic activity can minimize the high cytotoxic potential. In addition, they are among the biocatalysts most commercialized worldwide due to the versatility of reactions and substrates. However, although lipases are able to process cooking oil wastes, the products generated from this process do not necessarily become less toxic. Thus, the aim of the current study is to analyze the bioremediation of lipase-catalyzed cooking oil wastes, as well as their effect on the cytotoxicity of both the oil and its waste before and after enzymatic treatment. Thus, assessed the post-frying modification in soybean oil and in its waste, which was caused by hydrolysis reaction catalyzed by commercial and home-made lipases. The presence of lipases in the extracts obtained from orange wastes was identified by zymography. The profile of the fatty acid esters formed after these reactions was detected and quantified through gas chromatography and fatty acids profile compared through multivariate statistical analyses. Finally, the soybean oil and its waste, with and without enzymatic treatment, were assessed for toxicity in cytotoxicity assays conducted in vitro using fibroblast cell culture. The soybean oil wastes treated with core and frit lipases through transesterification reaction were less toxic than the untreated oils, thus confirming that cooking oil wastes can be bioremediated using orange lipases.