An Integrated In Vitro–In Silico Approach for Silver Nanoparticle Dosimetry in Cell Cultures

Potential human and environmental hazards resulting from the exposure of living organisms to silver nanoparticles (Ag NPs) have been the subject of intensive discussion in the last decade. Despite the growing use of Ag NPs in biomedical applications, a quantification of the toxic effects as a function of the total silver mass reaching cells (namely, target cell dose) is still needed. To provide a more accurate dose-response analysis, we propose a novel integrated approach combining well-established computational and experimental methodologies. We first used a particokinetic model (ISD3) for providing experimental validation of computed Ag NP sedimentation in static-cuvette experiments. After validation, ISD3 was employed to predict the total mass of silver reaching human endothelial cells and hepatocytes cultured in 96 well plates. Cell viability measured after 24 h of culture was then related to this target cell dose. Our results show that the dose perceived by the cell monolayer after 24 h of exposure is around 85% lower than the administered nominal media concentration. Therefore, accurate dosimetry considering particle characteristics and experimental conditions (e.g., time, size and shape of wells) should be employed for better interpreting effects induced by the amount of silver reaching cells

Proteomics Profiling of Heterozygous and Homozygous Patients with ABCA1 Gene Mutation: A Tangier Disease Molecular Map

Tangier Disease (TD) is a rare inherited disorder with approximately 100 worldwide identified cases. Alpha-lipoprotein deficiency is the main characteristic of this disease, associated with a virtual absence of High Density Lipoproteins (HDL) in blood. Additional symptoms are mild hypertriglyceridemia, neuropathy and enlarged, orange-colored tonsils. Genetically TD is caused by mutations in the ABCA1 gene, which prevent the release of cholesterol and phospholipids from cells, leading to the accumulation of lipids within cells and body tissues. In this work a TD patient and his parental heterozygous were examined from a proteomics point of view. Plasma as well as proteome and secretome of circulating monocytes were analyzed. Plasma proteins underlined in TD the imbalance of lipid trafficking and metabolism, associated with the stimulation of pro-inflammatory pathways. Proteome and secretome of monocytes highlighted an extensive down regulation of mitochondrial enzymes and vesicular trafficking agents along with a substantial cytoskeletal rearrangement, suggesting a reduced activation state of monocytes from TD homozygous patient. This work is the first proteomics profiling of heterozygous and homozygous TD phenotypes and it suggests a TD case as a model to understand general mechanisms of lipid transport and metabolism and their linkage to inflammatory processes

A proteomics approach to the study of bleomycin- induced lung fibrosis

Idiopathic pulmonary fibrosis (IPF) is the most severe lung fibrotic form and very few pharmacological therapies are available at present. Key events in the onset of the disease are the activation of fibroblasts to myofibroblasts and the production and release of extracellular matrix (ECM) and molecular factors. Primary murine lung fibroblasts were isolated and their activation induced by Bleomycin (BLM) treatment. Extracellular Vesicles (EV) were isolated and protein extracted. Released soluble proteins (Secretome) and EV-derived proteins were reduced, alkylated and trypsin digested. A nano-LC-MS/MS SWATHTM approach was used for the proteomics analyses. Specific proteins with a putative role in the transition from physiological to fibrotic conditions, such as several matrix metalloproteinases (MMPs), osteopontin (OPN), chitinase-3-like protein1 (CHI3L1) and CD44 resulted differentially released from BLM-treated fibroblasts as compared with untreated lung fibroblasts. Our results provide further understanding of the pathophysiological features of lung fibrosis, and suggest specific target for pharmacological treatments

RISC-mediated control of selected chromatin regulators stabilizes ground state pluripotency of mouse embryonic stem cells.

BACKGROUND: Embryonic stem cells are intrinsically unstable and differentiate spontaneously if they are not shielded from external stimuli. Although the nature of such instability is still controversial, growing evidence suggests that protein translation control may play a crucial role. RESULTS: We performed an integrated analysis of RNA and proteins at the transition between naïve embryonic stem cells and cells primed to differentiate. During this transition, mRNAs coding for chromatin regulators are specifically released from translational inhibition mediated by RNA-induced silencing complex (RISC). This suggests that, prior to differentiation, the propensity of embryonic stem cells to change their epigenetic status is hampered by RNA interference. The expression of these chromatin regulators is reinstated following acute inactivation of RISC and it correlates with loss of stemness markers and activation of early cell differentiation markers in treated embryonic stem cells. CONCLUSIONS: We propose that RISC-mediated inhibition of specific sets of chromatin regulators is a primary mechanism for preserving embryonic stem cell pluripotency while inhibiting the onset of embryonic developmental programs.This work was funded by: FIRB RBAP10L8TY (MIUR), Fondazione Roma and PAINCAGE FP7 Collaborative Project number 603191 (RB,MD); Flagship Project InterOmics PB.05 and MIUR-PRIN-2012 (FC); Wellcome Trust Core Grant reference 092096 and Cancer Research UK Grant Reference C6946/A14492 (LP); CRUK-Cambridge Institute Core Grant reference C14303/A17197 (DB)

A novel dynamic multicellular co-culture system for studying individual blood-brain barrier cell types in brain diseases and cytotoxicity testing

Blood brain barrier (BBB) cells play key roles in the physiology and pathology of the central nervous system (CNS). BBB dysfunction is implicated in many neurodegenerative diseases, including Alzheimer’s disease (AD). The BBB consists of capillary endothelial cells, pericytes encircling the endothelium and surrounding astrocytes extending their processes towards it. Although there have been many attempts to develop in vitro BBB models, the complex interaction between these celltypes makes it extremely difficult to determine their individual contribution to neurotoxicity in vivo. Thus, we developed and optimised an in vitro multicellular co-culture model within the Kirkstall Quasi Vivo System. The main aim was to determine the optimal environment to culture human brain primary endothelial cells, pericytes and astrocytes whilst maintaining cellular communication without formation of a barrier in order to assess the contribution of each cell type to the overall response. As a proof of concept for the present system, the effects of amyloid-beta 25-35 peptide (Aβ25-35), a hall mark of AD, were explored. This multicellular system will be a valuable tool for future studies on the specific roles of individual BBB cell type (while making connection with each other through medium) in CNS disorders as well as in cytotoxicity tests