Massimo Vassalli

Design and production of a chimeric resilin-, elastin-, and collagen-like engineered polypeptide.

Protein-inspired biomaterials have gained great interest as an alternative to synthetic polymers, in particular, for their potential use as biomedical devices. The potential inspiring models are mainly proteins able to confer mechanical properties to tissues and organs, such as elasticity (elastin, resilin, spider silk) and strength (collagen, silk). The proper combination of repetitive sequences, each of them derived from different proteins, represents a useful tool for obtaining biomaterials with tailored mechanical properties and biological functions. In this report we describe the design, the production, and the preliminary characterization of a chimeric polypeptide, based on sequences derived from the highly resilient proteins resilin and elastin and from collagen-like sequences. The results show that the obtained chimeric recombinant material exhibits promising self-assembling properties. Youngs modulus of the fibers was determined by AFM image analysis and lies in the range of 0.1-3 MPa in agreement with the expectations for elastin-like and resilin-like materials.

Surface nanoscale periodic structures in congruent lithium niobate by domain reversal patterning and differential etching

We report on the fabrication and characterization of one- and two-dimensional periodic structures down to 200 nm size, in congruent lithium niobate crystal samples. Periods from 2 mu m to 530 nm ...

Fluid viscosity determination by means of uncalibrated atomic force microscopy cantilevers

In this letter it has been proved that the vibrating resonance frequency of an atomic force microscope cantilever is strictly characterized by its thickness (α), while its width/thickness ratio (β) appears to be a less sensitive parameter that can be approximated to a constant. We therefore propose a data analysis method that, by accounting for a constant β, allows for the determination of the value of α and consequently to calculate η. This method of monitoring viscosity has the advantage of requiring short measurement times on very small sample volumes, thereby avoiding laborious, time-consuming cantilever calibration.

Electro-magnetic field promotes osteogenic differentiation of BM-hMSCs through a selective action on Ca2+-related mechanisms

Exposure to Pulsed Electromagnetic Field (PEMF) has been shown to affect proliferation and differentiation of human mesenchymal stem cells derived from bone marrow stroma (BM-hMSC). These cells offer considerable promise in the field of regenerative medicine, but their clinical application is hampered by major limitations such as poor availability and the time required to differentiate up to a stage suitable for implantation. For this reason, several research efforts are focusing on identifying strategies to speed up the differentiation process. In this work we investigated the in vitro effect of PEMF on Ca2+-related mechanisms promoting the osteogenic differentiation of BM-hMSC. Cells were daily exposed to PEMF while subjected to osteogenic differentiation and various Ca2+-related mechanisms were monitored using multiple approaches for identifying functional and structural modifications related to this process. The results indicate that PEMF exposure promotes chemically induced osteogenesis by mechanisms that mainly interfere with some of the calcium-related osteogenic pathways, such as permeation and regulation of cytosolic concentration, leaving others, such as extracellular deposition, unaffected. The PEMF effect is primarily associated to early enhancement of intracellular calcium concentration, which is proposed here as a reliable hallmark of the osteogenic developmental stage.

Effects of sphingosine 1-phosphate on excitation–contraction coupling in mammalian skeletal muscle

Sphingosine 1-phosphate (S1P) activates a subset of plasma membrane receptors of the endothelial differentiation gene family (EdgRs) in many cell types. In C2C12 myoblasts, exogenous S1P elicits Ca2+ transients by activating voltage-independent plasma membrane Ca2+ channels and intracellular Ca2+ -release channels. In this study, we investigated the effects of exogenous S1P on voltage-dependent L-type Ca2+ channels in skeletal muscle fibers from adult mice. To this end, intramembrane charge movements (ICM) and L-type Ca2+ current (ICa) were measured in single cut fibers using the double Vaseline-gap technique. Our data showed that submicromolar concentrations of S1P (100 nM) caused a ∼10-mV negative shift of the voltage threshold and transition voltages of qγ and qh components of ICM, and of ICa activation and inactivation. Biochemical studies showed that EdgRs are expressed in skeletal muscles. The involvement of EdgRs in the above S1P effects was tested with suramin, a specific inhibitor of Edg-3Rs. Suramin (200 μM) significantly reduced, by ∼90%, the effects of S1P on ICM and ICa, suggesting that most of S1P action occurred via Edg-3Rs. Moreover, S1P at concentration above 10 μM elicited intracellular Ca2+ transients in muscle fibers loaded with the fluorescent Ca2+ dye Fluo-3, as detected by confocal laser scanning microscopy.

Differential toxicity, conformation and morphology of typical initial aggregation states of Aβ1-42 and Aβpy3-42 beta-amyloids.

Among the different species of water-soluble β-peptides (Aβ1-42, Aβ1-40 and N-terminal truncated Aβ-peptides), Aβpy3-42 is thought to play a relevant role in Alzheimers pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Aβpy3-42, we separated different aggregation states of Aβ1-42 and Aβpy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates. Conformational analysis, by circular dichroism showed a prevailing random coil conformation for sa and ma, and typical β-sheet conformation for la. AFM and TEM show differential structural features between the three aggregates of a given β-peptide and among the aggregate of the two β-peptides. The potential toxic effects of the different aggregates were evaluated using human neuroblastoma SH-SY5Y cells in the MTT reduction, in the xCELLigence System, and in the Annexin V binding experiments. In the case of Aβ1-42 the most toxic aggregate is la, while in the case of Aβpy3-42 both sa and la are equally toxic. Aβ aggregates were found to be internalized in the cells, as estimated by confocal immunofluorescence microscopy, with a higher effect observed for Aβpy3-42, showing a good correlation with the toxic effects. Together these experiments allowed the discrimination of the intermediate states more responsible of oligomer toxicity, providing new insights on the correlation between the aggregation process and the toxicity and confirming the peculiar role in the pathogenesis of Alzheimer disease of Aβpy3-42 peptide.

THE HIGHLY CONSERVED SYNAPSIN DOMAIN E MEDIATES SYNAPSIN DIMERIZATION AND PHOSPHOLIPID VESICLE CLUSTERING

Synapsins are abundant SV (synaptic vesicle)-associated phosphoproteins that regulate synapse formation and function. The highly conserved C-terminal domain E was shown to contribute to several synapsin functions, ranging from formation of the SV reserve pool to regulation of the kinetics of exocytosis and SV cycling, although the molecular mechanisms underlying these effects are unknown. In the present study, we used a synthetic 25-mer peptide encompassing the most conserved region of domain E (Pep-E) to analyse the role of domain E in regulating the interactions between synapsin I and liposomes mimicking the phospholipid composition of SVs (SV-liposomes) and other pre-synaptic protein partners. In affinity-chromatography and cross-linking assays, Pep-E bound to endogenous and purified exogenous synapsin I and strongly inhibited synapsin dimerization, indicating a role in synapsin oligomerization. Consistently, Pep-E (but not its scrambled version) counteracted the ability of holo-synapsin I to bind and coat phospholipid membranes, as analysed by AFM (atomic force microscopy) topographical scanning, and significantly decreased the clustering of SV-liposomes induced by holo-synapsin I in FRET (Förster resonance energy transfer) assays, suggesting a causal relationship between synapsin oligomerization and vesicle clustering. Either Pep-E or a peptide derived from domain C was necessary and sufficient to inhibit both dimerization and vesicle clustering, indicating the participation of both domains in these activities of synapsin I. The results provide a molecular explanation for the effects of domain E in nerve terminal physiology and suggest that its effects on the size and integrity of SV pools are contributed by the regulation of synapsin dimerization and SV clustering.

Reconstructing the free-energy landscape of a polyprotein by single-molecule experiments

The mechanical unfolding of an engineered protein composed of eight domains of Ig27 is investigated by using atomic force microscopy. Exploiting a fluctuation relation, the equilibrium free energy as a function of the molecule elongation is estimated from pulling experiments. Such a free energy exhibits a regular shape that sets a typical unfolding length at zero force of the order of 20 nm. This length scale turns out to be much larger than the kinetic-unfolding length that is also estimated by analyzing the typical rupture force of the molecule under dynamic loading.

Role of the driving laser position on atomic force microscopy cantilevers excited by photothermal and radiation pressure effects

The excitation efficiency of the photothermal effect on coated microcantilevers has been studied for different flexural modes, both experimentally and theoretically, showing that the position of the driving laser is crucial to obtain a significant oscillation. Moreover, the characterization has been carried out on uncoated cantilevers, where the radiation pressure is not negligible with respect to the photothermal effect, showing that the laser position can be used to select which physical phenomenon is dominating the cantilever dynamics.

Sphingosine 1-phosphate induces cell contraction via calcium-independent/Rho-dependent pathways in undifferentiated skeletal muscle cells.

We have previously shown that sphingosine 1‐phosphate (S1P) can induce intracellular Ca2+ mobilization and cell contraction in C2C12 myoblasts and that the two phenomena are temporally unrelated. Although Ca2+‐independent mechanisms of cell contraction have been the focus of numerous studies on Ca2+ sensitization of smooth muscle, comparatively less studies have focused on the role that these mechanisms play in the regulation of skeletal muscle contractility. Phosphorylation and activation of myosin by Rho‐dependent kinase mediate most of Ca2+‐independent contractile responses. In the present study, we examined the potential role of Rho/Rho‐kinase cascade activation in S1P‐induced C2C12 cell contraction. First, we showed that depletion of Ca2+, by pre‐treatment with BAPTA, did not affect S1P‐induced myoblastic contractility, whereas it abolished S1P‐induced Ca2+ transients. These results correlated with the absence of troponin C and with the immature cytoskeletal organization of these cells. Experimental evidence demonstrating the involvement of Rho pathway in S1P‐stimulated myoblast contraction included: the activation/translocation of RhoA to the membrane in response to agonist‐stimulation in cells depleted of Ca2+ and the inhibition of dynamic changes of the actin cytoskeleton in cells where Rho functions had been inhibited either by overexpression of RhoGDI, a physiological inhibitor of GDP dissociation from Rho proteins, or by pretreatment with Y‐27632, a specific Rho kinase inhibitor. Contribution of protein kinase C in this cytoskeletal rearrangement was also evaluated. However, the pretreatment with Gö6976 or rottlerin, specific inhibitors of PKCα and PKCδ, respectively, failed to inhibit the agonist‐induced myoblastic contraction. Single particle tracking of G‐actin fluorescent probe was performed to statistically evaluate actin cytoskeletal dynamics in response to S1P. Stimulation with S1P was also able to increase the phosphorylation level of myosin light chain II. In conclusion, our results strongly suggest that Ca2+‐independent/Rho‐Rho kinase‐dependent pathways may exert an important role in S1P‐induced myoblastic cell contraction. J. Cell. Physiol. 198: 1–11, 2004© 2003 Wiley‐Liss, Inc.