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Rota P.,University of Milan | Anastasia L.,University of Milan | Anastasia L.,Laboratory of Stem Cells for Tissue Engineering | Allevi P.,University of Milan
Organic and Biomolecular Chemistry | Year: 2015

The current analytical protocol used for the GC-MS determination of free or 1,7-lactonized natural sialic acids (Sias), as heptafluorobutyrates, overlooks several transformations. Using authentic reference standards and by combining GC-MS and NMR analyses, flaws in the analytical protocol were pinpointed and elucidated, thus establishing the scope and limitations of the method. It was demonstrated that (a) Sias 1,7-lactones, even if present in biological samples, decompose under the acidic hydrolysis conditions used for their release; (b) Sias 1,7-lactones are unpredicted artifacts, accidentally generated from their parent acids; (c) the N-acetyl group is quantitatively exchanged with that of the derivatizing perfluorinated anhydride; (d) the partial or complete failure of the Sias esterification-step with diazomethane leads to the incorrect quantification and structure attribution of all free Sias. While these findings prompt an urgent correction and improvement of the current analytical protocol, they could be instrumental for a critical revision of many incorrect claims reported in the literature. This journal is © The Royal Society of Chemistry 2015. Source

Verdelli C.,Laboratory of Molecular Biology | Avagliano L.,University of Milan | Creo P.,Laboratory of Stem Cells for Tissue Engineering | Guarnieri V.,Medical Genetics | And 9 more authors.
Endocrine-Related Cancer | Year: 2015

Components of the tumour microenvironment initiate and promote cancer development. In this study, we investigated the stromal component of parathyroid neoplasia. Immuno-histochemistry for alpha-smooth muscle actin (α-SMA) showed an abundant periacinar distribution of α-SMA+ cells in normal parathyroid glands (nZ3). This pattern was progressively lost in parathyroid adenomas (PAds; nZ6) where α-SMA+ cells were found to surround new microvessels, as observed in foetal parathyroid glands (nZ2). Moreover, in atypical adenomas (nZ5) and carcinomas (nZ4), α-SMA+ cells disappeared from the parenchyma and accumulated in the capsula and fibrous bands. At variance with normal glands, parathyroid tumours (nZ37) expressed high levels of fibroblast-activation protein (FAP) transcripts, a marker of tumour-associated fibroblasts. We analysed the ability of PAd-derived cells to activate fibroblasts using human bone-marrow mesenchymal stem cells (hBM-MSCs). PAd-derived cells induced a significant increase in FAP and vascular endothelial growth factor A (VEGFA) mRNA levels in co-cultured hBM-MSCs. Furthermore, the role of the calcium-sensing receptor (CASR) and of the CXCL12/CXCR4 pathway in the PAd-induced activation of hBM-MSCs was investigated. Treatment of co-cultures of hBM-MSCs and PAd-derived cells with the CXCR4 inhibitor AMD3100 reduced the stimulated VEGFA levels, while CASR activation by the R568 agonist was ineffective. PAd-derived cells co-expressing parathyroid hormone (PTH)/CXCR4 and PTH/CXCL12 were identified by FACS, suggesting a paracrine/autocrine signalling. Finally, CXCR4 blockade by AMD3100 reduced PTH gene expression levels in PAd-derived cells. In conclusion, i) PAd-derived cells activated cells of mesenchymal origin; ii) PAd-associated fibroblasts were involved in tumuor neoangiogenesis and iii) CXCL12/CXCR4 pathway was expressed and active in PAd cells, likely contributing to parathyroid tumour neoangiogenesis and PTH synthesis modulation. © 2015 Society for Endocrinology. Source

Bugiardini E.,University of Milan | Rivolta I.,University of Milan Bicocca | Binda A.,University of Milan Bicocca | Soriano Caminero A.,Penn State Hershey Medical Center | And 8 more authors.
Neuromuscular Disorders | Year: 2015

In myotonic dystrophy type 2 (DM2), an association has been reported between early and severe myotonia and recessive chloride channel (. CLCN1) mutations. No DM2 cases have been described with sodium channel gene (. SCN4A) mutations. The aim is to describe a DM2 patient with severe and early onset myotonia and co-occurrence of a novel missense mutation in SNC4A. A 26-year-old patient complaining of hand cramps and difficulty relaxing her hands after activity was evaluated at our department. Neurophysiology and genetic analysis for DM1, DM2, CLCN1 and SCN4A mutations were performed. Genetic testing was positive for DM2 (2650 CCTG repeat) and for a variant c.215C>T (p.Pro72Leu) in the SCN4A gene. The variation affects the cytoplasmic N terminus domain of Nav1.4, where mutations have never been reported. The biophysical properties of the mutant Nav1.4 channels were evaluated by whole-cell voltage-clamp analysis of heterologously expressed mutant channel in tsA201 cells. Electrophysiological studies of the P72L variant showed a hyperpolarizing shift (-5mV) of the voltage dependence of activation that may increase cell excitability. This case suggests that SCN4A mutations may enhance the myotonic phenotype of DM2 patients and should be screened for atypical cases with severe myotonia. © 2015 Elsevier B.V.. Source

Massaccesi L.,University of Milan | Burlina A.,University of Padua | Baquero C.J.,University of Milan | Goi G.,University of Milan | And 2 more authors.
Clinical Biochemistry | Year: 2011

Objectives: ERT application to Fabry's disease patients needs sensitive assay method of the missing enzyme (α-d-galactosidase A) to achieve early diagnosis. Design and methods: A new fluorimetric assay method of alpha;-d-galactosidase A was developed, using whole blood (WB) from 30 healthy individuals, 7 hemizygous males and 7 heterozygous females with Fabry's disease. This method was compared with the traditional dried blood spot (DBS) method. Results: WB method analytical characteristics are: linearity up to 2000. mU/L; detection limit: 4. mU/L; linearity versus time: 6. h; enzyme stability: 7. days at 4 °C; total analytical imprecision: from 3.27% to 5.72%. Sensitivity was higher in WB than DBS method. All hemizygous Fabry's patients were identified by both the WB and DBS methods. With regards to the seven heterozygous carriers five could be identified by the WB methods and three by the DBS method. Conclusion: The WB assay method for α-d-galactosidase A appears to be reliable and proposable as a routine method for prompt diagnosis of Fabry disease in selected at-risk populations. © 2011 The Canadian Society of Clinical Chemists. Source

Piccoli M.,Laboratory of Stem Cells for Tissue Engineering | Cirillo F.,Laboratory of Stem Cells for Tissue Engineering | Tettamanti G.,Laboratory of Stem Cells for Tissue Engineering | Anastasia L.,Laboratory of Stem Cells for Tissue Engineering | Anastasia L.,University of Milan
European Heart Journal, Supplement | Year: 2016

The possibility of generating induced pluripotent stem cells from mouse embryonic fibroblasts and human adult fibroblasts has introduced new perspectives for possible therapeutic strategies to repair damaged hearts. However, obtaining large numbers of adult stem cells is still an ongoing challenge, and the safety of genetic reprogramming with lenti- or retro-viruses has several drawbacks not easy to be addressed. Furthermore, the majority of adult stem cell-based clinical trials for heart regeneration have had generally poor and controversial results. Nonetheless, it is now clear that the injected cells activate the growth and differentiation of progenitor cells that are already present in the heart. This is achieved by the release of signalling factors and/or exosomes carrying them. Along this line, chemistry may play a major role in developing new strategies for activating resident stem cells to regenerate the heart. In particular, this review focuses on small molecule approaches for cell reprogramming, cell differentiation, and activation of cell protection. © 2016 Published on behalf of the European Society of Cardiology. All rights reserved. Source

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