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.

CoCr-enriched medium modulates integrin-based downstream signaling and requires a set of inflammatory genes reprograming in vitro.

Significant health concerns have been raised by the high levels of Cr and Co ions into whole blood as resulted of corrosion process released from biomedical implants, but very little is known about their biological behavior in governing cell metabolism. Thus, we prompted to address this issue by exploring the effects of CoCr enriched medium on both fibroblast and preosteoblast (pre-Ob) cells. First, we showed there is a significant difference in Co and Cr releasing dependent on engineered surface, it being even more released in dual acid-etching treating surface (named w/DAE) than the machined surfaces (named wo/DAE). Thereafter, we showed CoCr affects pre-osteoblast and fibroblast metabolism by dynamically modulating integrin-based downstream signaling (FAK, Src, Rac1, and Cofilin). Specifically on this matter, we have shown there is dynamic ß1-integrin gene activation up 24 h in both preosteoblast and fibroblast. Our analysis showed also that both pre-Ob and fibroblast are important resource of proinflammatory cytokines when responding to CoCr enriched medium. In addition, survival-related signaling pathway was also affected interfering on survival and proliferating signal, mainly affecting CDK2, mapk-Erk and mapk-p38 phosphorylations, while AKT/PKB-related gene remained active. In addition, during cell adhesion PP2A (an important Ser/Thr phosphatase) was inactive in both cell lineages and it seems be a CoCr's molecular fingerprint, regulating specific metabolic pathways involved with cytoskeleton rearrangement. Altogether, our results showed for the first time CoCr affects cellular performance in vitro by modulating integrin activation-based downstream signaling and requiring a reprograming of inflammatory genes activations in vitro. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 839-849, 2018.

GFAAS determination of mercury in muscle samples of fish from Amazon, Brazil.

In the present study, a simple, rapid and sensitive method was developed for the determination of mercury concentrations in the muscle tissue of fish from the Brazilian Amazon using graphite furnace atomic absorption spectrometry (GFAAS) following acid mineralization of the samples in an ultrasonic cold water bath. Using copper nitrate as a chemical modifier in solution and sodium tungstate as permanent modifier, we were able to attain thermal stabilization of the mercury up to the atomisation temperature of 1600 °C in the GFAAS assay. The calculated limits of detection (LOD) and quantification (LOQ) were 0.014 and 0.047 mg kg(-1), respectively.