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Pastorino F.,Experimental Therapy Unit | Simone G.,Italian Institute of Technology
17th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2013

We present a novel microdroplet-based device for extensive characterization of carbohydrate-protein complexes in a single experiment with online analysis. Microdroplets compartmentalized the carbohydrates and proteins. Specific interaction promoted complex formation and the complexes emitted localized signal. The dissociation constant KD was achieved by quantification of the fluorescent signal and Michaelis-Menten model fitting. The analyzed carbohydrate was tested in neuroblastoma (NB) cell lines. The results show a solid approach to quantify the sensitivity of the glyco-assay. Source

Martinengo C.,University of Turin | Martinengo C.,Center for Experimental Research and Medical Studies | Poggio T.,University of Turin | Poggio T.,Center for Experimental Research and Medical Studies | And 23 more authors.
Cancer Research

Rearrangements involving the anaplastic lymphoma kinase (ALK) gene are defining events in several tumors, including anaplastic large-cell lymphoma (ALCL) and non - small cell lung carcinoma (NSCLC). In such cancers, the oncogenic activity of ALK stimulates signaling pathways that induce cell transformation and promote tumor growth. In search for common pathways activated by oncogenic ALK across different tumors types, we found that hypoxia pathways were significantly enriched in ALK-rearranged ALCL and NSCLC, as compared with other types of T-cell lymphoma or EGFR- and K- RAS-mutated NSCLC, respectively. Consistently, in both ALCL and NSCLC, we found that under hypoxic conditions, ALK directly regulated the abundance of hypoxia-inducible factors (HIF), which are key players of the hypoxia response in normal tissues and cancers. In ALCL, the upregulation of HIF1α and HIF2α in hypoxic conditions required ALK activity and its downstream signaling proteins STAT3 and C/EBPβ. In vivo, ALK regulated VEGFA production and tumor angiogenesis in ALCL and NSCLC, and the treatment with the anti-VEGFA antibody bevacizumab strongly impaired ALCL growth in mouse xenografts. Finally, HIF2α, but not HIF1α, was required for ALCL growth in vivo whereas the growth and metastasis potential of ALK-rearranged NSCLC required both HIF1α and HIF2α. In conclusion, we uncovered an ALK-specific regulation of the hypoxia response across different ALK+ tumor types and propose HIFs as a powerful speci fic therapeutic target in ALK-rearranged ALCL and NSCLC. ©2014 AACR. Source

Di Paolo D.,Experimental Therapy Unit | Brignole C.,Experimental Therapy Unit | Pastorino F.,Experimental Therapy Unit | Carosio R.,Experimental Therapy Unit | And 11 more authors.
Molecular Therapy

RNA interference molecules have some advantages as cancer therapeutics, including a proved efficacy on both wild-type (WT) and mutated transcripts and an extremely high sequence-specificity. The most significant hurdle to be overcome if exogenous small interfering RNAs (siRNA) is to be used therapeutically is the specific, effective, nontoxic delivery of siRNA to its intracellular site of action. At present, human applications are confined almost exclusively to targets within the liver, where the delivery systems naturally accumulate, and extra-hepatic targets remain a challenge. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that has recently been shown to contribute to the cell growth and progression of human neuroblastoma (NB). We investigated its potential as a therapeutic target in NB by generating anti-GD 2-targeted nanoparticles that carry ALK-directed siRNA, which are specifically and efficiently delivered to GD 2-expressing NB cells. Relative to free ALK-siRNA, anti-GD 2-targeted liposomal formulations of ALK-siRNA had low plasma clearance, increased siRNA stability, and improved binding, uptake, silencing and induction of cell death, and specificity for NB cells. In NB xenografts, intravenous (i.v.) injection of the targeted ALK-siRNA liposomes showed gene-specific antitumor activity with no side effects. ALK-selective siRNA entrapped in anti-GD 2-targeted nanoparticles is a promising new modality for NB treatment. © The American Society of Gene &Cell Therapy. Source

Wilson C.L.,Northumbria University | Mann J.,Northumbria University | Walsh M.,Northumbria University | Perrugoria M.J.,Northumbria University | And 8 more authors.

Toll-like Receptor 3 (TLR3) is a pathogen pattern recognition receptor that plays a key role in innate immunity. TLR3 signalling has numerous functions in liver, both in health and disease. Here we report that TLR3 is expressed by quiescent hepatic stellate cells (HSC) where it functions to induce transcription and secretion of functional interferons as well as a number of other cytokines and chemokines. Upon transdifferentiation into myofibroblasts, HSCs rapidly loose the ability to produce interferon gamma (IFNγ). Mechanistically, this gene silencing may be due to Polycomb complex mediated repression via methylation of histone H3 lysine 27. In contrast to wild type, quiescent HSC isolated from tlr3 knockout mice do not produce IFNγ in response to Poly(I:C) treatment. Therefore, quiescent HSC may contribute to induction of the hepatic innate immune system in response to injury or infection. © 2014 Wilson et al. Source

Curnis F.,San Raffaele Scientific Institute | Gasparri A.M.,San Raffaele Scientific Institute | Longhi R.,CNR Institute of Chemistry of Molecular Recognition | Colombo B.,San Raffaele Scientific Institute | And 4 more authors.
Cellular and Molecular Life Sciences

Chromogranin A (CgA), a secretory protein expressed by many neuroendocrine cells, neurons, cardiomyocytes, and keratinocytes, is the precursor of various peptides that regulate the carbohydrate/lipid metabolism and the cardiovascular system. We have found that CgA, locally administered to injured mice, can accelerate keratinocyte proliferation and wound healing. This biological activity was abolished by the Asp 45Glu mutation. CgA and its N-terminal fragments, but not the corresponding Asp 45Glu mutants, could selectively recognize the αvβ6-integrin on keratinocytes (a cell-adhesion receptor that is up-regulated during wound healing) and regulate keratinocyte adhesion, proliferation, and migration. No binding was observed to other integrins such as αvβ3, αvβ5, αvβ8, α5β1, α1β1, α3β1, α6β4, α6β7 and α9β1. Structure-activity studies showed that the entire CgA 39-63 region is crucial for avb6 recognition (Ki = 7 nM). This region contains an RGD site (residues CgA 43-45) followed by an amphipathic α-helix (residues CgA 47-63), both crucial for binding affinity and selectivity. These results suggest that the interaction of the RGD/a-helix motif of CgA with αvβ6 regulates keratinocyte physiology in wound healing. © 2012 Springer Basel AG. Source

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