Papers by Roberto Raiteri
Computer Methods in Biomechanics and Biomedical Engineering, Aug 18, 2018
Articular cartilage is a tissue of fundamental importance for the mechanics of joints, since it p... more Articular cartilage is a tissue of fundamental importance for the mechanics of joints, since it provides a smooth and lubricated surface for the proper transfer of loads. From a mechanical point of view, this tissue is an anisotropic poroviscoelastic material: its characteristics at the macroscopic level depend on the complex microscopic architecture. With the ability to probe the local microscopic features, dynamic nanoindentation test is a powerful tool to investigate cartilage mechanics. In this work we focus on a length scale where the time dependent behaviour is regulated by poroelasticity more than viscoelasticity and we aim to understand the effect of the anisotropic permeability on the mechanics of the superficial layer of the articular cartilage. In a previous work, a finite element model for the dynamic nanoindentation test has been presented. In this work, we improve the model by considering the presence of an anisotropic permeability tensor that depends on the collagen fibers distribution. Our sensitivity analysis highlights that the permeability decreases with increasing indentation, thus making the tissue stiffer than the case of isotropic permeability, when solicited at the same frequency. With this improved model, a revised identification of the mechanical and physical parameters for articular cartilage is provided. To this purpose the model was used to simulate experimental data from tests performed on bovine tissue, giving a better estimation of the anisotropy in the elastic properties. A relation between the identified macroscopic anisotropic permeability properties and the microscopic rearrangement of the fiber/matrix structure during indentation is also provided.
Biophysical Journal, 2012
The physiological function of calmodulin (CaM) depends on its ability to respond specifically to ... more The physiological function of calmodulin (CaM) depends on its ability to respond specifically to micromolar Ca 2þ signals in the presence of ~1000-fold excess of the chemically similar Mg 2þ . The intracellular concentration of Mg 2þ is tightly controlled in a narrow range of 0.5-1.0 mM, which is sufficient to fully or partially saturate the Ca 2þ -binding sites of many EF-hand proteins, including CaM, at the resting Ca 2þ levels. To explain the mechanism of metal ion binding specificity we have compared the effects of Mg 2þ (ionic radius r=0.72 A ˚), Mn 2þ (r=0.67 A ˚) and Ca 2þ (r=1.06 A ˚) on the structure and stability of the N-terminal domain of calmodulin (N-CaM). The far UV-CD melting profiles show that all three metal ions stabilize the structure of N-CaM, as indicated by the concentration dependent increase in unfolding temperature from 52 C (apo) to >100 C, 95 C and 75 C in the presence of Ca 2þ , Mn 2þ and Mg 2þ , respectively. We have determined the X-ray structures of N-CaM complexed with the three metal ions. Only the Ca 2þ -N-CaM complex features the opendomain active conformation, whereas Mg 2þ -N-CaM and Mn 2þ -N-CaM have a closed-domain, apo-like conformation. The relative positions of all bound metal ions with respect to the EF-hand-b-scaffold are similar, however the bidentate ligand, the Glu sidechain in the 12 th position of the loop, interacts with Ca 2þ only and does not bind directly to either Mn 2þ or Mg 2þ . These results strongly support our proposal (Grabarek, BBA, 1813, 913, 2011) that the stereochemical constraints imposed by the two-EF-hand domain structure preclude Mg 2þ from engaging the EF-hand ligands in the same way as Ca 2þ . Mg 2þ stabilizes the apo-like conformation of an EF-hand, thus contributing to the switching off CaM activity at the resting Ca 2þ concentrations. Lobe-Specific Functions of Calcium.Calmodulin in Alpha-Calcium.
Journal of Neural Engineering, Apr 5, 2018
Nature Communications, Oct 8, 2015
In striated muscle, X-ROS is the mechanotransduction pathway by which mechanical stress transduce... more In striated muscle, X-ROS is the mechanotransduction pathway by which mechanical stress transduced by the microtubule network elicits reactive oxygen species. X-ROS tunes Ca 2 þ signalling in healthy muscle, but in diseases such as Duchenne muscular dystrophy (DMD), microtubule alterations drive elevated X-ROS, disrupting Ca 2 þ homeostasis and impairing function. Here we show that detyrosination, a post-translational modification of a-tubulin, influences X-ROS signalling, contraction speed and cytoskeletal mechanics. In the mdx mouse model of DMD, the pharmacological reduction of detyrosination in vitro ablates aberrant X-ROS and Ca 2 þ signalling, and in vivo it protects against hallmarks of DMD, including workload-induced arrhythmias and contraction-induced injury in skeletal muscle. We conclude that detyrosinated microtubules increase cytoskeletal stiffness and mechanotransduction in striated muscle and that targeting this post-translational modification may have broad therapeutic potential in muscular dystrophies.
Biophysical Journal, Feb 1, 2016
Ryanodine receptors (RyRs) control the release of intracellular calcium from sarcoplasmic reticul... more Ryanodine receptors (RyRs) control the release of intracellular calcium from sarcoplasmic reticulum and play a central role in excitation-contraction coupling of skeletal and cardiac muscle. Many modulatory proteins and small molecules interact and regulate the function of RyR, including FKBP12 (FK506 binding proteins of 12), a protein ligand that binds RyR with nanomolar affinity. FKBP12 stabilizes the closed state of the channel and reduces the appearance of subconductance states. Dissociation of FKBP12 can cause the calcium leak from RyR and has been implicated in several disorders, such as muscular dystrophy, sarcopenia, heart failure, diabetes, or Alzheimer's. Previous studies using cryo-EM and FRET suggest FKBP12 binds to the periphery of cytoplasmic domain, but the exact binding determinants within the RyR protein at the single amino acid level had remained unclear. The recent high-resolution cryo-EM studies also disagree on the identity of the RyR domain interacting with FKBP12. We determined the high-resolution crystal structures of several RyR domains. Computational docking of these structures, combined with FRET trilateration experiments, show that SPRY1 domain is located next to FKBP12. The key residues in SPRY1 critical for the binding of FKBP12 were identified by molecular dynamics flexible fitting and verified by mutagenesis and functional assays. Disease mutations in SPRY1 were investigated by x-ray crystallography. One of them directly affects FKBP12 binding properties by interfering domain folding. This structural model provides a valuable template for designing therapeutic drugs targeting RyR-FKBP12 interface to treat the diseases mentioned above.
Materials, Jul 24, 2021
We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic... more We have developed a novel experimental set-up that simultaneously, (i) applies static and dynamic deformations to adherent cells in culture, (ii) allows the visualization of cells under fluorescence microscopy, and (iii) allows atomic force microscopy nanoindentation measurements of the mechanical properties of the cells. The cell stretcher device relies on a dielectric elastomer film that can be electro-actuated and acts as the cell culture substrate. The shape and position of the electrodes actuating the film can be controlled by design in order to obtain specific deformations across the cell culture chamber. By using optical markers we characterized the strain fields under different electrode configurations and applied potentials. The combined setup, which includes the cell stretcher device, an atomic force microscope, and an inverted optical microscope, can assess in situ and with sub-micron spatial resolution single cell topography and elasticity, as well as ion fluxes, during the application of static deformations. Proof of performance on fibroblasts shows a reproducible increase in the average cell elastic modulus as a response to applied uniaxial stretch of just 4%. Additionally, high resolution topography and elasticity maps on a single fibroblast can be acquired while the cell is deformed, providing evidence of long-term instrumental stability. This study provides a proof-of-concept of a novel platform that allows in situ and real time investigation of single cell mechano-transduction phenomena with sub-cellular spatial resolution.
Applied sciences, Dec 22, 2022
Over the past 10 years, the number of dental implants has grown significantly. This increase has ... more Over the past 10 years, the number of dental implants has grown significantly. This increase has consequently led to an elevation of the statistics related to cases of peri-implantitis. Laser therapy has conquered a place among the therapies of excellence to treat peri-implantitis. However, the laser device used could influence the therapy's success. The aim of this comparative experimental work was to highlight the differences in the work on grade 4 titanium surfaces of the most commonly used laser lights in this field, taking into consideration any structural damage that lasers could cause to implant surfaces. The lasers examined were a 980 nm diode laser; a 1064 nm Nd:YAG laser; and a new generation of 1064 nm Nd:YAG Q-switch nano pulsed laser. We evaluated the titanium temperature increase, the pre-and post-treatment two-dimensional surface appearance observed under the scanning electron microscope; finally, the three-dimensional pre-and post-treatment topographic analysis was assessed using atomic force microscopy. We showed that the 1064-nm Q-switch Nd:YAG nanosecond pulsed laser appears to be more suitable for the preservation of implant morphology because of the absence of the induction of metal damage.
Medical Engineering & Physics, Jul 1, 2014
arXiv (Cornell University), Mar 22, 2000
In this work we investigate the influence of the use of YSZ and CeO2/YSZ as insulators for Metal-... more In this work we investigate the influence of the use of YSZ and CeO2/YSZ as insulators for Metal-Ferroelectric-Insulator-Semiconductor (MFIS) structures made with SrBi2Ta2O9 (SBT). We show that by using YSZ only the a-axis oriented Pyrochlore phase could be obtained. On the other hand the use of a CeO2/YSZ double-buffer layer gave a c-axis oriented SBT with no amorphous SiO2 inter-diffusion layer. The characteristics of MFIS diodes were greatly improved by the use of the double buffer. Using the same deposition conditions the memory window could be increased from 0.3 V to 0.9 V. From the piezoelectric response, nano-meter scale ferroelectric domains could be clearly identified in SBT thin films.
Biophysical Journal, 2015
Ionic strength (IS) is an important parameter to govern the inter-and intramolecular interactions... more Ionic strength (IS) is an important parameter to govern the inter-and intramolecular interactions. In muscle, it has been known that an increase in IS lowers Ca 2þ activated tension, however, its molecular mechanism is not well understood. Our aim was to determine whether force/cross-bridge or the number of force-generating cross-bridges changes with IS. Stiffness during rigor was studied on single fibers from rabbit psoas, which showed that there was no effect of IS, demonstrating that in-series compliance is not affected by IS. This observation indicates that stiffness of thick filament, thin filament, myosin head, and actomyosin interface are not affected by IS. Sinusoidal analyses were performed during Ca 2þ activation, and the effects of ATP, phosphate (Pi), and ADP on three rate constants were studied at IS ranging 150mM-300mM to characterize elementary steps of the cross-bridge cycle. Both ATP binding (K1) and ADP binding (K0) increased to 2x, and the Pi binding (K5) decreased to 1/2 when IS was increased from 150mM to 300mM. The effect of Pi can be explained by the electrostatic interaction with the Pi binding site on myosin. The effect on ATP/ADP can be attributed to improved stereoscopic and hydrophobic interaction with the nucleotide binding site. The increase in IS increased cross-bridge detachment steps (k2 and k(-4)), indicating that electrostatic force counteracts these steps. However, IS did not affect attachment steps (k(-2) and k4). Consequently, the equilibrium constant of the detachment step (K2) increased to 2x, and the force generation step (K4) decreased to 0.7x. These effects together diminished the number of force-generating cross-bridges by ~10%. Because associated decrease of tension was ~40%, the major effect of IS is a decrease in force/cross-bridge, but also a decrease in the number of force generating cross-bridge occurs.
Journal of Zhejiang University-science B, Sep 1, 2006
We report an important observation that the surface conductivity of antibody layer immobilized on... more We report an important observation that the surface conductivity of antibody layer immobilized on polylysine-coated glass substrate decreases upon the formation of complex with their specific antigens. This change in conductivity has been observed for both monoclonal and polyclonal antibodies. The conductance of monoclonal mouse IgG immobilized on polylysine-coated glass substrate changed from 1.02×10 -8 Ω -1 to 1.41×10 -11 Ω -1 at 10 V when complex is formed due to the specific biomolecular interactions with rabbit anti-mouse IgG F(ab′) 2 . Similar behavior was observed when the same set up was tested in two clinical assays: (1) anti-Leishmania antigen polyclonal antibodies taken from Kala Azar positive patient serum interacting with Leishmania promastigote antigen, and (2) anti-p21 polyclonal antibodies interacting with p21 antigen. The proposed concept can represent a new immunodiagnostic technique and may have wide ranging applications in biosensors and nanobiotechnology too.
Applied Sciences
Over the past 10 years, the number of dental implants has grown significantly. This increase has ... more Over the past 10 years, the number of dental implants has grown significantly. This increase has consequently led to an elevation of the statistics related to cases of peri-implantitis. Laser therapy has conquered a place among the therapies of excellence to treat peri-implantitis. However, the laser device used could influence the therapy’s success. The aim of this comparative experimental work was to highlight the differences in the work on grade 4 titanium surfaces of the most commonly used laser lights in this field, taking into consideration any structural damage that lasers could cause to implant surfaces. The lasers examined were a 980 nm diode laser; a 1064 nm Nd:YAG laser; and a new generation of 1064 nm Nd:YAG Q-switch nano pulsed laser. We evaluated the titanium temperature increase, the pre- and post-treatment two-dimensional surface appearance observed under the scanning electron microscope; finally, the three-dimensional pre- and post-treatment topographic analysis was...
Sensors and Actuators B: Chemical, 2018
Biomaterials, Feb 1, 2018
The availability of 3D biomimetic in vitro neuronal networks of mammalian neurons represents a pi... more The availability of 3D biomimetic in vitro neuronal networks of mammalian neurons represents a pivotal step for the development of brain-on-a-chip experimental models to study neuronal (dys)functions and particularly neuronal connectivity. The use of hydrogel-based scaffolds for 3D cell cultures has been extensively studied in the last years. However, limited work on biomimetic 3D neuronal cultures has been carried out to date. In this respect, here we investigated the use of a widely popular polysaccharide, chitosan (CHI), for the fabrication of a microbead based 3D scaffold to be coupled to primary neuronal cells. CHI microbeads were characterized by optical and atomic force microscopies. The cell/scaffold interaction was deeply characterized by transmission electron microscopy and by immunocytochemistry using confocal microscopy. Finally, a preliminary electrophysiological characterization by micro-electrode arrays was carried out.
Experimental dermatology, 2018
Skin mechanical properties are usually measured considering the entire skin thickness and very li... more Skin mechanical properties are usually measured considering the entire skin thickness and very little is known about the mechanical behaviour of individual skin layers. We propose atomic force microscopy (AFM) as a tool to quantify nanoscale changes in the biomechanical properties and ultrastructure of human papillary dermis exposed to different mechanical and physical stimuli. Samples from 3 human skin biopsies were studied: one stretched by obesity, one subjected to a high level of sun exposure and normal skin as control. Slices of the papillary dermis layer were harvested at controlled depths from each skin biopsy and 25 μm areas of each slice were imaged and D-periodicity of collagen fibres measured by AFM, together with their stiffness. Standard histological analysis was also carried out to correlate biochemical properties and their distribution with stiffness and topography. We obtained similar stiffness values between the sample affected by obesity and the control sample at a...
The Journal of Physical Chemistry, 1996
The aim of this study was to measure interaction forces between surfaces with high electric poten... more The aim of this study was to measure interaction forces between surfaces with high electric potentials in aqueous electrolyte solutions. Therefore, the force between a platinum or gold sample, which served as the working electrode, and a silicon nitride tip of an atomic force microscope was measured. Various potentials were applied between the sample and a reference electrode. Experimental results were compared to forces calculated with the Poisson-Boltzmann equation. As predicted by theory, the electrostatic double-layer force changed only in a narrow potential range of about 300 mV and saturated below and above this range. Within this range the repulsion grew with more negative sample potentials. This was expected, since the tip was negatively charged at the high pH chosen. At strong negative sample potentials this saturation was not complete and the force continued to rise slightly when lowering the potential. Another surprising and yet unexplained observation was a weak long-range attraction at positive sample potentials. This attraction decayed with a decay length of typically 50 nm. In parallel, the structure of Au(111) was imaged. We confirmed a ( 3 × p, p > 10) reconstruction at potentials below about -0.3 V SHE and the normal (1 × 1) hexagonal packing above this potential. Above about +0.8 V SHE the (1 × 1) structure disappeared and no crystalline packing was observed anymore.
2010 Annual International Conference of the Ieee Engineering in Medicine and Biology Society, 2010
The Oriented architecture of macromolecules plays a critical role in many aspects of Nanobiotechn... more The Oriented architecture of macromolecules plays a critical role in many aspects of Nanobiotechnology such as in the development of biosensors. To this regard, S-layers which constitute the outermost cell envelope component of many prokaryotic organisms, represent unique self assembled systems with the capability to rearrange into monomolecular and oriented arrays. These properties can be exploited to promote their crystallization on surfaces (e.g. silicone) which is pivotal for the subsequent immobilization of macromolecules and development of new biosensors. In this work the crystallization of bacterial S-layers obtained from Bacillus thuringiensis and Bacillus sphaericus CCM2177 on silicone, mica and quartz crystal surfaces were investigated. The SDS page results of S-layers isolated from the above mentioned bacteria put in evidence that their molecular weight (MW) was around 120 KDa and, as reported in the literature, slightly higher for those extracted by Bacillus thuringiensis. In addition, results showed that S-layers isolated from Bacillus thuringiensis form large crystalline domains on mica after 5 min whereas those extracted from Bacillus sphaericus CCM 2177 form a compact monolayer on silicone after 2 h. Results in this work put in evidence the possibility to use these substrates for the fabrication of sensitive biosensors.
Sensors and Actuators B: Chemical, 2001
The merging of silicon microfabrication techniques with surface functionalization biochemistry of... more The merging of silicon microfabrication techniques with surface functionalization biochemistry offers new exciting opportunities in developing microscopic biomedical analysis devices with unique characteristics. Micro-mechanical transducers for chemical and biosensing applications represent one possibility. Microcantilevers can transduce a chemical signal into a mechanical motion with high sensitivity. In this review we summarize how cantilever-based sensors can be operated, and their working principle is presented in few selected biosensing experiments which have been performed recently in our groups in the study of biotin±streptavidin and antigen±antibody interactions, and speci®c surface charge development of organic molecules. We also discuss the advantages of this novel technique as well as its potentials.
Sensors and Actuators B: Chemical, 1998
The authors measured electrostatic interaction between the silicon nitride tip of an atomic force... more The authors measured electrostatic interaction between the silicon nitride tip of an atomic force microscope (AFM) and different silicon insulators surfaces: silicon oxide; the silicon nitride layer of a light addressable potentiometric sensor (LAPS) and the silicon nitride surface of an ion sensitive field effect transistor (ISFET) device produced by a modified process with an additional annealing step, immersed in
Review of Scientific Instruments, 2001
Interparticle and surface forces are of great importance in many fields of pure and applied scien... more Interparticle and surface forces are of great importance in many fields of pure and applied science. We present an apparatus to measure the normal and friction forces acting between a particle (radius of 0.5–20 μm) and another solid surface. The apparatus is based on the principle of an atomic force microscope. For quantitative friction measurements we propose a method to determine the lateral spring constants of atomic force microscope cantilevers with attached spherical particles.
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Papers by Roberto Raiteri