New insights in osteogenic differentiation revealed by mass spectrometric assessment of phosphorylated substrates in murine skin mesenchymal cells

Abstract Background Bone fractures and loss represent significant costs for the public health system and often affect the patients quality of life, therefore, understanding the molecular basis for bone regeneration is essential. Cytokines, such as IL-6, IL-10 and TNFα, secreted by inflammatory cells at the lesion site, at the very beginning of the repair process, act as chemotactic factors for mesenchymal stem cells, which proliferate and differentiate into osteoblasts through the autocrine and paracrine action of bone morphogenetic proteins (BMPs), mainly BMP-2. Although it is known that BMP-2 binds to ActRI/BMPR and activates the SMAD 1/5/8 downstream effectors, little is known about the intracellular mechanisms participating in osteoblastic differentiation. We assessed differences in the phosphorylation status of different cellular proteins upon BMP-2 osteogenic induction of isolated murine skin mesenchymal stem cells using Triplex Stable Isotope Dimethyl Labeling coupled with LC/MS. Results From 150 μg of starting material, 2,264 proteins were identified and quantified at five different time points, 235 of which are differentially phosphorylated. Kinase motif analysis showed that several substrates display phosphorylation sites for Casein Kinase, p38, CDK and JNK. Gene ontology analysis showed an increase in biological processes related with signaling and differentiation at early time points after BMP2 induction. Moreover, proteins involved in cytoskeleton rearrangement, Wnt and Ras pathways were found to be differentially phosphorylated during all timepoints studied. Conclusions Taken together, these data, allow new insights on the intracellular substrates which are phosphorylated early on during differentiation to BMP2-driven osteoblastic differentiation of skin-derived mesenchymal stem cells.

Prevalence and risk factors of Hepatitis C virus infection in Brazil, 2005 through 2009: a cross-sectional study

Abstract Background Hepatitis C chronic liver disease is a major cause of liver transplant in developed countries. This article reports the first nationwide population-based survey conducted to estimate the seroprevalence of HCV antibodies and associated risk factors in the urban population of Brazil. Methods The cross sectional study was conducted in all Brazilian macro-regions from 2005 to 2009, as a stratified multistage cluster sample of 19,503 inhabitants aged between 10 and 69 years, representing individuals living in all 26 State capitals and the Federal District. Hepatitis C antibodies were detected by a third-generation enzyme immunoassay. Seropositive individuals were retested by Polymerase Chain Reaction and genotyped. Adjusted prevalence was estimated by macro-regions. Potential risk factors associated with HCV infection were assessed by calculating the crude and adjusted odds ratios, 95% confidence intervals (95% CI) and p values. Population attributable risk was estimated for multiple factors using a case–control approach. Results The overall weighted prevalence of hepatitis C antibodies was 1.38% (95% CI: 1.12%–1.64%). Prevalence of infection increased in older groups but was similar for both sexes. The multivariate model showed the following to be predictors of HCV infection: age, injected drug use (OR = 6.65), sniffed drug use (OR = 2.59), hospitalization (OR = 1.90), groups socially deprived by the lack of sewage disposal (OR = 2.53), and injection with glass syringe (OR = 1.52, with a borderline p value). The genotypes 1 (subtypes 1a, 1b), 2b and 3a were identified. The estimated population attributable risk for the ensemble of risk factors was 40%. Approximately 1.3 million individuals would be expected to be anti-HCV-positive in the country. Conclusions The large estimated absolute numbers of infected individuals reveals the burden of the disease in the near future, giving rise to costs for the health care system and society at large. The known risk factors explain less than 50% of the infected cases, limiting the prevention strategies. Our findings regarding risk behaviors associated with HCV infection showed that there is still room for improving strategies for reducing transmission among drug users and nosocomial infection, as well as a need for specific prevention and control strategies targeting individuals living in poverty.