Proteomic Analysis Reveals a Mitochondrial Remodeling of βTC3 Cells in Response to Nanotopography

Recently, using cluster-assembled zirconia substrates with tailored roughness produced by supersonic cluster beam deposition, we demonstrated that \u3b2 cells can sense nanoscale features of the substrate and can translate these stimuli into a mechanotransductive pathway capable of preserveing \u3b2-cell differentiation and function in vitro in long-term cultures of human islets. Using the same proteomic approach, we now focused on the mitochondrial fraction of \u3b2TC3 cells grown on the same zirconia substrates and characterized the morphological and proteomic modifications induced by the nanostructure. The results suggest that, in \u3b2TC3 cells, mitochondria are perturbed by the nanotopography and activate a program involving metabolism modification and modulation of their interplay with other organelles. Data were confirmed in INS1E, a different \u3b2-cell model. The change induced by the nanostructure can be pro-survival and prime mitochondria for a metabolic switch to match the new cell needs

MosChito rafts as effective and eco-friendly tool for the delivery of a Bacillus thuringiensis-based insecticide to Aedes albopictus larvae

Adult mosquito females, through their bites, are responsible for the transmission of different zoonotic pathogens. Although adult control represents a pillar for the prevention of disease spread, larval control is also crucial. Herein we characterized the effectiveness of a suitable tool, named "MosChito raft", for the aquatic delivery of a Bacillus thuringiensis var. israelensis (Bti) formulate, a bioinsecticide active by ingestion against mosquito larvae. MosChito raft is a floating tool composed by chitosan cross-linked with genipin in which a Bti-based formulate and an attractant have been included. MosChito rafts (i) resulted attractive for the larvae of the Asian tiger mosquito Aedes albopictus, (ii) induced larval mortality within a few hours of exposure and, more importantly, (iii) protected the Bti-based formulate, whose insecticidal activity was maintained for more than one month in comparison to the few days residual activity of the commercial product. The delivery method was effective in both laboratory and semi-field conditions, demonstrating that MosChito rafts may represent an original, eco-based and user-friendly solution for larval control in domestic and peri-domestic aquatic habitats such as saucers and artificial containers in residential or urban environments

Proteomic dissection of nanotopography-sensitive mechanotransductive signaling hubs that foster neuronal differentiation in PC12 cells

Neuronal cells are competent in precisely sensing nanotopographical features of their microenvironment. The perceived microenvironmental information will be \ue2\u80\u9cinterpreted\ue2\u80\u9d by mechanotransductive processes and impacts on neuronal functioning and differentiation. Attempts to influence neuronal differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the fact that profound details of mechanosensing/-transduction complexity remain elusive. Introducing omics methods into these biomaterial approaches has the potential to provide a deeper insight into the molecular processes and signaling cascades underlying mechanosensing/-transduction but their exigence in cellular material is often opposed by technical limitations of major substrate top-down fabrication methods. Supersonic cluster beam deposition (SCBD) allows instead the bottom-up fabrication of nanostructured substrates over large areas characterized by a quantitatively controllable ECM-like nanoroughness that has been recently shown to foster neuron differentiation and maturation. Exploiting this capacity of SCBD, we challenged mechanosensing/-transduction and differentiative behavior of neuron-like PC12 cells with diverse nanotopographies and/or changes of their biomechanical status, and analyzed their phosphoproteomic profiles in these settings. Versatile proteins that can be associated to significant processes along the mechanotransductive signal sequence, i.e., cell/cell interaction, glycocalyx and ECM, membrane/f-actin linkage and integrin activation, cell/substrate interaction, integrin adhesion complex, actomyosin organization/cellular mechanics, nuclear organization, and transcriptional regulation, were affected. The phosphoproteomic data suggested furthermore an involvement of ILK, mTOR, Wnt, and calciumsignaling in these nanotopography- and/or cell mechanics-related processes. Altogether, potential nanotopography-sensitive mechanotransductive signaling hubs participating in neuronal differentiation were dissected

Scale invariant disordered nanotopography promotes hippocampal neuron development and maturation with involvement of mechanotransductive pathways

The identification of biomaterials which promote neuronal maturation up to the generation of integrated neural circuits is fundamental for modern neuroscience. The development of neural circuits arises from complex maturative processes regulated by poorly understood signaling events, often guided by the extracellular matrix (ECM). Here we report that nanostructured zirconia surfaces, produced by supersonic cluster beam deposition of zirconia nanoparticles and characterized by ECM-like nanotopographical features, can direct the maturation of neural networks. Hippocampal neurons cultured on such cluster-assembled surfaces displayed enhanced differentiation paralleled by functional changes. The latter was demonstrated by single-cell electrophysiology showing earlier action potential generation and increased spontaneous postsynaptic currents compared to the neurons grown on the featureless unnaturally flat standard control surfaces. Label-free shotgun proteomics broadly confirmed the functional changes and suggests furthermore a vast impact of the neuron/nanotopography interaction on mechanotransductive machinery components, known to control physiological in vivo ECM-regulated axon guidance and synaptic plasticity. Our results indicate a potential of cluster-assembled zirconia nanotopography exploitable for the creation of efficient neural tissue interfaces and cell culture devices promoting neurogenic events, but also for unveiling mechanotransductive aspects of neuronal development and maturation

Growth Mechanism of Cluster-Assembled Surfaces : From Submonolayer to Thin-Film Regime

Nanostructured films obtained by assembling preformed atomic clusters are of strategic importance for a wide variety of applications. The deposition of clusters produced in the gas phase onto a substrate offers the possibility to control and engineer the structural and functional properties of the cluster-assembled films. To date, the microscopic mechanisms underlying the growth and structuring of cluster-assembled films are poorly understood, and, in particular, the transition from the submonolayer to the thin-film regime is experimentally unexplored. Here we report the systematic characterization by atomic force microscopy of the evolution of the structural properties of cluster-assembled films deposited by supersonic cluster beam deposition. As a paradigm of nanostructured systems, we focus our attention on cluster-assembled zirconia films, investigating the influence of the building block dimensions on the growth mechanisms and roughening of the thin films, following the growth process from the early stages of the submonolayer to the thin-film regime. Our results demonstrate that the growth dynamics in the submonolayer regime determines different morphological properties of the cluster-assembled thin film. The evolution of the roughness with the number of deposited clusters reproduces the growth exponent of the ballistic deposition in the 2 thorn 1 model from the submonolayer to the thin-film regime

Adhesion force spectroscopy with nanostructured colloidal probes reveals nanotopography-dependent early mechanotransductive interactions at the cell membrane level

Mechanosensing, the ability of cells to perceive and interpret the microenvironmental biophysical cues (such as the nanotopography), impacts strongly cellular behaviour through mechanotransductive processes and signalling. These events are predominantly mediated by integrins, the principal cellular adhesion receptors located at the cell/extracellular matrix (ECM) interface. Because of the typical piconewton force range and nanometre length scale of mechanotransductive interactions, achieving a detailed understanding of the spatiotemporal dynamics occurring at the cell/microenvironment interface is challenging; sophisticated interdisciplinary methodologies are required. Moreover, an accurate control over the nanotopographical features of the microenvironment is essential, in order to systematically investigate and precisely assess the influence of the different nanotopographical motifs on the mechanotransductive process. In this framework, we were able to study and quantify the impact of microenvironmental nanotopography on early cellular adhesion events by means of adhesion force spectroscopy based on innovative colloidal probes mimicking the nanotopography of natural ECMs. These probes provided the opportunity to detect nanotopography-specific modulations of the molecular clutch force loading dynamics and integrin clustering at the level of single binding events, in the critical time window of nascent adhesion formation. Following this approach, we found that the nanotopographical features are responsible for an excessive force loading in single adhesion sites after 20-60 s of interaction, causing a drop in the number of adhesion sites. However, by manganese treatment we demonstrated that the availability of activated integrins is a critical regulatory factor for these nanotopography-dependent dynamics

Tribological coatings for complex mechanical elements produced by supersonic cluster beam deposition of metal dichalcogenide nanoparticles

Fullerene-like MoS2 and WS2 nanoparticles can be used as building blocks for the fabrication of fluid and solid lubricants. Metal dichalcogenide films have a very low friction coefficient in vacuum, therefore they have mostly been used as solid lubricants in space and vacuum applications. Unfortunately, their use is significantly hampered by the fact that in the presence of humidity, oxygen and moisture, the low-friction properties of these materials rapidly degrade due to oxidation. The use of closed-cage MoS2 and WS2 nanoparticles may eliminate this problem, although the fabrication of lubricant thin films starting from dichalcogenide nanoparticles is, to date, a difficult task. Here we demonstrate the use of supersonic cluster beam deposition for the coating of complex mechanical elements (angular contact ball bearings) with nanostructured MoS2 and WS2 thin films. We report structural and tribological characterization of the coatings in view of the optimization of tribological performances for aerospace applications

An Amperometric Sensor for Thiocholine Based on Cluster-Assembled Zirconia Modified Electrodes

Here we report on the fabrication and characterization of cluster-assembled nanostructured zirconia electrodes for the electrochemical detection of enzymatically produced thiocholine. Zirconia nanostructures are produced by Supersonic Cluster Beam Deposition on thin gold films. This technique enables nanoscale control of the deposited film surface morphology, providing high active surface area for electrochemical detection of the analyte, along with high double-layer capacitance and suitable charge transfer resistance of the system. The electrochemical behavior of the electrodes has been characterized in the presence of the Potassium ferricyanide/Potassium ferrocyanide redox couple and the system performance showed to be enhanced starting from a thickness of the deposited layer of 60 nm. The electrochemical response for the oxidation of an enzymatic product was assessed by means of cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry. The nanostructured zirconia film yields a good electrochemical detection of thiocholine. The limit of detection for thiocholine under working potential of 0.810-0.820 V versus reference was found to be comprised between 0.25 mu M and 1.3 mu M. Nanostructured electrodes, combining gold and zirconia nanoparticles can be implemented as functional transducers in biosensing devices, for example based on Acetylcholinesterase for electrochemical detection of polluting agents