Centrosome amplification mediates small extracellular vesicles secretion via lysosome disruption

PreprintSummary Bidirectional communication between cells and their surrounding environment is critical in both normal and pathological settings. Extracellular vesicles (EVs), which facilitate the horizontal transfer of molecules between cells, are recognized as an important constituent of cell-cell communication. In cancer, alterations in EV secretion contribute to the growth and metastasis of tumor cells. However, the mechanisms underlying these changes remain largely unknown. Here, we show that centrosome amplification is associated with and sufficient to promote small extracellular vesicle ( S EV) secretion in pancreatic cancer cells. This is a direct result due of lysosomal dysfunction, caused by increased reactive oxygen species (ROS) downstream of extra centrosomes. Defects in lysosome function promotes multivesicular body fusion with the plasma membrane, thereby enhancing S EV secretion. Furthermore, we find that S EVs secreted in response to amplified centrosomes are functionally distinct and activate pancreatic stellate cells (PSCs). These activated PSCs promote the invasion of pancreatic cancer cells in heterotypic 3-D cultures. We propose that S EVs secreted by cancer cells with amplified centrosomes influence the bidirectional communication between the tumor cells and the surrounding stroma to promote malignancy

Centrosome amplification mediates small extracellular vesicle secretion via lysosome disruption

International audienc

Detection of food-borne pathogens with DNA arrays on disk

A DNA oligonucleotide array for duplex pathogen detection on a DVD platform is developed. The assay involves hybridization of PCR products and optical detection using compact disc technology. Different DNA array constructions for attachment of synthetic oligonucleotides on to DVD surface are evaluated, finding that streptavidin-biotin coupling method yielded the highest sensitivity in combination with enzymatic signal amplification. Issues of importance for the DNA array construction such immobilized probes design, PCR product labeling strategy and composition of the hybridization buffer were addressed. The methodology was proved scoring single nucleotide polymorphisms with high selectivity. The assay capability was also demonstrated by the identification of two pathogenic microorganisms in powder milk samples. In fifty minutes, the DVD-array system identifies Salmonella spp. and Cronobacter spp. (previously named Enterobacter sakazakii) precise and simultaneously with a sensitivity of 100 and 102 cfu/mL, respectively, in infant milk. Results were in good agreement with those obtained by quantitative real-time PCR. © 2012 Elsevier B.V. All rights reserved.This work was funded by the projects PATSENS, PSE-010000-2008-6 (Spanish Government and EU FEDER funds), FEDER CTQ2010-15943 (CICYT, Spain), PROMETEO 2010/008 and ACOMP/2012/158 (Generalitat Valenciana). The Spanish MEC provided T.A-C with a grant for her PhD studies.Arnandis Chover, T.; Morais, S.; Tortajada-Genaro, LA.; Puchades, R.; Maquieira Catala, Á.; Berganza, J.; Olabarria, G. (2012). Detection of food-borne pathogens with DNA arrays on disk. Talanta. 101:405-412. https://doi.org/10.1016/j.talanta.2012.09.049S40541210

Inhibition of Calpain Prevents Manganese-Induced Cell Injury and Alpha-Synuclein Oligomerization in Organotypic Brain Slice Cultures

Overexposure to manganese has been known to promote alpha-synuclein oligomerization and enhance cellular toxicity. However, the exact mechanism of Mn-induced alpha-synuclein oligomerization is unclear. To explore whether alpha-synuclein oligomerization was associated with the cleavage of alpha-synuclein by calpain, we made a rat brain slice model of manganism and pretreated slices with calpain inhibitor II, a cell-permeable peptide that restricts the activity of calpain. After slices were treated with 400 μM Mn for 24 h, there were significant increases in the percentage of apoptotic cells, lactate dehydrogenase release, intracellular [Ca2+]i, calpain activity, and the mRNA and protein expression of calpain 1 and alpha-synuclein. Moreover, the number of C- and N-terminal fragments of alpha-synuclein and the amount of alpha-synuclein oligomerization also increased. These results also showed that calpain inhibitor II pretreatment could reduce Mn-induced nerve cell injury and alpha-synuclein oligomerization. Additionally, there was a significant decrease in the number of C- and N-terminal fragments of alpha-synuclein in calpain inhibitor II-pretreated slices. These findings revealed that Mn induced the cleavage of alpha-synuclein protein via overactivation of calpain and subsequent alpha-synuclein oligomerization in cultured slices. Moreover, the cleavage of alpha-synuclein by calpain 1 is an important signaling event in Mn-induced alpha-synuclein oligomerization