Reliability based robustness of timber structures through NDT data updating

This work presents a framework for reliability-based assessment of timber structures / members using data gathered from non-destructive test results. These results are used for modeling an update of the mechanical characteristics of timber, using Bayesian methods. These methods are suitable to be used for parameter estimation and also allow updating model uncertainties. From the updated model, decisions upon the life-cycle reliability of existing structures may be taken and maintenance or strengthening actions may be considered. In this work, results gathered from ultrasound testing, Resistograph® and Pilodyn® conducted on chestnut wood specimens were used, as well as correlations between those results and compression strength parallel to the grain tests’ results. The resistant characteristics are also updated assuming deterioration models applied to specific key elements of the structure, thus, being possible to evaluate reliability based in time dependent factors, as well to categorize that structure in terms of robustness.COST E55, STSM-6269Fundação de Ciência e Tecnologia, FCT, through project SFRH/BD/62326/2009

The VAR2CSA malaria protein efficiently retrieves circulating tumor cells in an EpCAM-independent manner

Isolation of metastatic circulating tumor cells (CTCs) from cancer patients is of high value for disease monitoring and molecular characterization. Despite the development of many new CTC isolation platforms in the last decade, their isolation and detection has remained a challenge due to the lack of specific and sensitive markers. In this feasibility study, we present a method for CTC isolation based on the specific binding of the malaria rVAR2 protein to oncofetal chondroitin sulfate (ofCS). We show that rVAR2 efficiently captures CTCs from hepatic, lung, pancreatic, and prostate carcinoma patients with minimal contamination of peripheral blood mononuclear cells. Expression of ofCS is present on epithelial and mesenchymal cancer cells and is equally preserved during epithelial-mesenchymal transition of cancer cells. In 25 stage I-IV prostate cancer patient samples, CTC enumeration significantly correlates with disease stage. Lastly, rVAR2 targets a larger and more diverse population of CTCs compared to anti-EpCAM strategies

Failure of human rhombic lip differentiation underlies medulloblastoma formation

Medulloblastoma (MB) comprises a group of heterogeneous paediatric embryonal neoplasms of the hindbrain with strong links to early development of the hindbrain 1–4. Mutations that activate Sonic hedgehog signalling lead to Sonic hedgehog MB in the upper rhombic lip (RL) granule cell lineage 5–8. By contrast, mutations that activate WNT signalling lead to WNT MB in the lower RL 9,10. However, little is known about the more commonly occurring group 4 (G4) MB, which is thought to arise in the unipolar brush cell lineage 3,4. Here we demonstrate that somatic mutations that cause G4 MB converge on the core binding factor alpha (CBFA) complex and mutually exclusive alterations that affect CBFA2T2, CBFA2T3, PRDM6, UTX and OTX2. CBFA2T2 is expressed early in the progenitor cells of the cerebellar RL subventricular zone in Homo sapiens, and G4 MB transcriptionally resembles these progenitors but are stalled in developmental time. Knockdown of OTX2 in model systems relieves this differentiation blockade, which allows MB cells to spontaneously proceed along normal developmental differentiation trajectories. The specific nature of the split human RL, which is destined to generate most of the neurons in the human brain, and its high level of susceptible EOMES +KI67 + unipolar brush cell progenitor cells probably predisposes our species to the development of G4 MB

Extending the Compatibility of the SP3 Paramagnetic Bead Processing Approach for Proteomics

The diversity in protein and peptide biochemistry necessitates robust protocols and reagents for efficiently handling and enriching these molecules prior to analysis with mass spectrometry (MS) or other techniques. Further exploration of the paramagnetic bead-based approach, single-pot solid-phase-enhanced sample preparation (SP3), is carried out toward updating and extending previously described conditions and experimental workflows. The SP3 approach was tested in a wide range of experimental scenarios, including (1) binding solvents (acetonitrile, ethanol, isopropanol, acetone), (2) binding pH (acidic vs neutral), (3) solvent/lysate ratios (50–200%, v/v), (4) mixing and rinsing conditions (on-rack vs off-rack rinsing), (5) Enrichment of nondenatured proteins, and (6) capture of individual proteins from noncomplex mixtures. These results highlight the robust handling of proteins in a broad set of scenarios while also enabling the development of a modified SP3 workflow that offers extended compatibility. The modified SP3 approach is used in quantitative in-depth proteome analyses to compare it with commercial paramagnetic bead-based HILIC methods (MagReSyn) and across multiple binding conditions (e.g., pH and solvent during binding). Together, these data reveal the extensive quantitative coverage of the proteome possible with SP3 independent of the binding approach utilized. The results further establish the utility of SP3 for the unbiased handling of peptides and proteins for proteomic applications

Extending the Compatibility of the SP3 Paramagnetic Bead Processing Approach for Proteomics

The diversity in protein and peptide biochemistry necessitates robust protocols and reagents for efficiently handling and enriching these molecules prior to analysis with mass spectrometry (MS) or other techniques. Further exploration of the paramagnetic bead-based approach, single-pot solid-phase-enhanced sample preparation (SP3), is carried out toward updating and extending previously described conditions and experimental workflows. The SP3 approach was tested in a wide range of experimental scenarios, including (1) binding solvents (acetonitrile, ethanol, isopropanol, acetone), (2) binding pH (acidic vs neutral), (3) solvent/lysate ratios (50–200%, v/v), (4) mixing and rinsing conditions (on-rack vs off-rack rinsing), (5) Enrichment of nondenatured proteins, and (6) capture of individual proteins from noncomplex mixtures. These results highlight the robust handling of proteins in a broad set of scenarios while also enabling the development of a modified SP3 workflow that offers extended compatibility. The modified SP3 approach is used in quantitative in-depth proteome analyses to compare it with commercial paramagnetic bead-based HILIC methods (MagReSyn) and across multiple binding conditions (e.g., pH and solvent during binding). Together, these data reveal the extensive quantitative coverage of the proteome possible with SP3 independent of the binding approach utilized. The results further establish the utility of SP3 for the unbiased handling of peptides and proteins for proteomic applications

α-Synuclein pathology in Parkinson disease activates homeostatic NRF2 anti-oxidant response

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleinopathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2/Nrf2) is recognized as ‘the master regulator of cellular anti-oxidant response’, both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on the serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, expressing the mutant human A53T aSyn), which manifests widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.Medicine, Faculty ofNon UBCPathology and Laboratory Medicine, Department ofReviewedFacult

The Insulin-Like Growth Factor I Receptor Is Required for Akt Activation and Suppression of Anoikis in Cells Transformed by the ETV6-NTRK3 Chimeric Tyrosine Kinase

Signaling through the insulin-like growth factor I receptor (IGF-IR) axis is essential for transformation by many dominantly acting oncoproteins. However, the mechanism by which IGF-IR contributes to oncogenesis remains unknown. To examine this, we compared transformation properties of the oncogenic ETV6-NTRK3 (EN) chimeric tyrosine kinase in IGF-IR-null R(−) mouse embryo fibroblasts with R(−) cells engineered to reexpress IGF-IR (R(+) cells). We previously showed that R(−) cells expressing EN (R(−) EN cells) are resistant to transformation but that transformation is restored in R(+) cells. We now show that while R(−) EN cells have intact Ras-extracellular signal-regulated kinase signaling and cell cycle progression, they are defective in phosphatidylinositol-3-kinase (PI3K)-Akt activation and undergo detachment-induced apoptosis (anoikis) under anchorage-independent conditions. In contrast, R(+) cells expressing EN (R(+) EN cells) suppress anoikis and are fully transformed. The requirement for IGF-IR in R(−) EN cells is overcome by ectopic expression of either activated Akt or a membrane-targeted form of EN. Moreover, compared to R(−) EN cells, R(+) EN cells show a dramatic increase in membrane localization of insulin receptor substrate 1 (IRS-1) in association with EN. Since EN is known to bind IRS-1 as an adaptor protein, our findings suggest that IGF-IR may function to localize EN/IRS-1 complexes to cell membranes, in turn facilitating PI3K-Akt activation and suppression of anoikis