Arthroscopic Treatment of a Subchondral Bone Cyst via Stem Cells Application: A Case Study in Equine Model and Outcomes.

Subchondral bone cysts in horses represent one of the main causes of lameness that can occur in different anatomical locations. The study describes the treatment in regenerative therapy of the intracystic implantation of adipose tissue mesenchymal stromal cells (AMSCs) included in platelet-rich plasma (PRP). The ability of AMSCs to differentiate in osteogenic cells was tested in vitro and in vivo.

Innovative Nanostructured Fillers for Dental Resins: Nanoporous Alumina and Titania Nanotubes.

The possibility of improving dental restorative materials is investigated through the addition of two different types of fillers to a polymeric resin. These fillers, consisting of porous alumina and TiO nanotubes, are compared based on their common physicochemical properties on the nanometric scale. The aim was to characterize and compare the surface morphological properties of composite resins with different types of fillers using analytical techniques.

Articular Cartilage Regeneration by Hyaline Chondrocytes: A Case Study in Equine Model and Outcomes.

Cartilage injury defects in animals and humans result in the development of osteoarthritis and the progression of joint deterioration. Cell isolation from equine hyaline cartilage and evaluation of their ability to repair equine joint cartilage injuries establish a new experimental protocol for an alternative approach to osteochondral lesions treatment. Chondrocytes (CCs), isolated from the autologous cartilage of the trachea, grown in the laboratory, and subsequently arthroscopically implanted into the lesion site, were used to regenerate a chondral lesion of the carpal joint of a horse.

Mesoscale Ordering of Phycocyanin Molecules in Langmuir-Blodgett Multilayers.

Phycocyanin molecules, which are part of light-harvesting complexes in cyanobacteria, can be assembled into mesoscale multilayer nanofilms by the Langmuir-Blodgett technique. Results obtained by quartz crystal microbalance and atomic force microscopy confirm the homogeneity and reproducibility of phycocyanin Langmuir-Blodgett multilayer deposition. We show by cryo-electron microdiffraction that amorphous phycocyanin Langmuir-Blodgett multilayers form, after annealing at 150 °C and cooling to room temperature, a layered nanofibrillar lattice with rotational disorder.

Langmuir-Blodgett nanotemplates for protein crystallography.

The new generation of synchrotrons and microfocused beamlines has enabled great progress in X-ray protein crystallography, resulting in new 3D atomic structures for proteins of high interest to the pharmaceutical industry and life sciences. It is, however, often still challenging to produce protein crystals of sufficient size and quality (order, intensity of diffraction, radiation stability). In this protocol, we provide instructions for performing the Langmuir-Blodgett (LB) nanotemplate method, a crystallization approach that can be used for any protein (including membrane proteins).

Langmuir-Blodgett (LB)-based nanobiocrystallography at the frontiers of cancer proteomics.

Background/aim: Langmuir-Blodgett (LB) films used as templates for crystallization lead to marked changes in protein stability and water dehydration, despite slight changes in protein atomic structure. Herein, we discuss the importance of LB-based nanocrystallography at the frontiers of cancer proteomics focusing on two model proteins with important biological roles in cancer, namely CK2alpha and RNase A.

Materials And Methods: Computational mutagenesis using the KINARI Mutagen webserver exhibits different behaviors in terms of stability and robustness, as well as in terms of water dynamics.

A review of the strategies for obtaining high-quality crystals utilizing nanotechnologies and microgravity.

Crystallization is a highly demanding and time-consuming task that causes a real bottle-neck in basic research. Great effort has been made to understand the factors and parameters that influence this process and to finely tune them to facilitate crystal growth. Different crystallization techniques have been proposed over the past decades, such as the classical vapor hanging drop method, its variant the sitting drop method, dialysis, cryo-temperature, gel, batch, and the innovative microgravity (space) techniques like free interface diffusion (FID) and counter-ion diffusion (CID).

Identification of best protein crystallization methods by molecular dynamics (MD).

A full-atom structure of a protein provides an important piece of information for molecular biologists, but has to be complemented by further knowledge concerning its conformational mobility and functional properties. Some scholars have proposed to integrate proteomics-derived data (mainly obtained with techniques like X-ray and NMR crystallography) with protein bioinformatics and computational approaches, above all molecular dynamics (MD), in order to gain better elucidations about proteins. MD simulations have been applied to different areas of protein sciences, but so far few efforts have been made to couple MD with an understanding of the different crystallization techniques that have been proposed during the decades, like classical vapor diffusion hanging drop and its variants (such as sitting drop), in space- and LB (Langmuir-Blodgett)-based crystallization procedures.

Advances in nanocrystallography as a proteomic tool.

In order to overcome the difficulties and hurdles too much often encountered in crystallizing a protein with the conventional techniques, our group has introduced the innovative Langmuir-Blodgett (LB)-based crystallization, as a major advance in the field of both structural and functional proteomics, thus pioneering the emerging field of the so-called nanocrystallography or nanobiocrystallography. This approach uniquely combines protein crystallography and nanotechnologies within an integrated, coherent framework that allows one to obtain highly stable protein crystals and to fully characterize them at a nano- and subnanoscale. A variety of experimental techniques and theoretical/semi-theoretical approaches, ranging from atomic force microscopy, circular dichroism, Raman spectroscopy and other spectroscopic methods, microbeam grazing-incidence small-angle X-ray scattering to in silico simulations, bioinformatics, and molecular dynamics, has been exploited in order to study the LB-films and to investigate the kinetics and the main features of LB-grown crystals.

Proteomics and proteogenomics approaches for oral diseases.

Design and implementation of new biocompatible materials and achievements in the field of nanogenomics and nanoproteomics as well as in other related and allied sciences in the broader framework of translational and clinical nanomedicine are paving new avenues for nanodentistry. Classical dentistry is becoming more predictive, preventive, personalized, and participatory, providing the patients with a tailored and targeted treatment and handling of their diseases. Considering the global impact of the oral pathologies, being particularly heavy in underdeveloped and developing countries, it is mandatory from an ethical perspective to ensure a global oral health.

Conductometric monitoring of protein-protein interactions.

Conductometric monitoring of protein-protein and protein-sterol interactions is here proved feasible by coupling quartz crystal microbalance with dissipation monitoring (QCM_D) to nucleic acid programmable protein arrays (NAPPA). The conductance curves measured in NAPPA microarrays printed on quartz surface allowed the identification of binding events between the immobilized proteins and the query. NAPPA allows the immobilization on the quartz surface of a wide range of proteins and can be easily adapted to generate innumerous types of biosensors.

Atomic force microscopy and anodic porous allumina of nucleic acid programmable protein arrays.

The methodological aspects are here presented for the NAPPA (Nucleic Acid Programmable Protein Arrays) characterization by atomic force microscopy and anodic porous alumina. Anodic Porous Alumina represents also an advanced on chip laboratory for gene expression contained in an engineered plasmid vector. The results obtained with CdK2, CDKN1A, p53 and Jun test genes expressed on NAPPA and the future developments are discussed in terms of our pertinent and recent Patents and of their possibility to overcome some limitations of present fluorescence detection in probing protein-protein interaction in both basic sciences and clinical studies.

Prototypes of newly conceived inorganic and biological sensors for health and environmental applications.

This paper describes the optimal implementation of three newly conceived sensors for both health and environmental applications, utilizing a wide range of detection methods and complex nanocomposites. The first one is inorganic and based on matrices of calcium oxide, the second is based on protein arrays and a third one is based on Langmuir-Blodgett laccase multi-layers. Special attention was paid to detecting substances significant to the environment (such as carbon dioxide) and medicine (drug administration, cancer diagnosis and prognosis) by means of amperometric, quartz crystal microbalance with frequency (QCM_F) and quartz crystal microbalance with dissipation monitoring (QCM_D) technologies.

Langmuir-Blodgett nanotemplate and radiation resistance in protein crystals: state of the art.

A state-of-the-art review of the role of the Langmuir-Blodgett nanotemplate on protein crystal structures is here presented. Crystals grown by nanostructured template appear more radiation resistant than the classical ones, even in the presence of a third-generation highly focused beam at the European Synchrotron Radiation Facility. The electron density maps and the changes in parameters such as total diffractive power, B-factor, and pairwise R-factor have been discussed.

Recombinant laccase: II. Medical biosensor.

Langmuir-Blodgett (LB) technology was used to build a high-sensitivity enzyme-based biosensor for medical purposes. Recombinant fungal laccase from Rigidoporous lignosus, as previously described, was used to catalyze a widely used antidepressant in a micromolar range, namely, clomipramine. The topological properties of the laccase thin film were characterized via LB π-A isotherm and AFM (mean roughness 8.

Recombinant laccase: I. Enzyme cloning and characterization.

We obtained structural and functional characterization of a recombinant Laccase from Rigidoporus lignosus (formerly Rigidoporus microporus), a white-rot basidiomycete, by means of circular dichroism (CD) spectra, cyclic voltammetry (CV) and biochemical assays. Here we report the optimization of expression and purification procedures of a recombinant Laccase expressed in supercompetent Escherichia coli cells. We amplified the coding sequence of Laccase using PCR from cDNA and cloned into a bacterial expression system.

Nanoproteomics enabling personalized nanomedicine.

Nucleic Acid Programmable Protein Arrays utilize a complex mammalian cell free expression system to produce proteins in situ. In alternative to fluorescent-labeled approaches a new label free method, emerging from the combined utilization of three independent and complementary nanotechnological approaches, appears capable to analyze protein function and protein-protein interaction in studies promising for personalized medicine. Quartz Micro Circuit nanogravimetry, based on frequency and dissipation factor, mass spectrometry and anodic porous alumina overcomes indeed the limits of correlated fluorescence detection plagued by the background still present after extensive washes.

Unique water distribution of Langmuir-Blodgett versus classical crystals.

Langmuir-Blodgett films when used as nanotemplates for crystallization often leads to marked changes in protein stability and structure. Earlier we found that stability of proteins is also correlated with aqueous surroundings in the crystals. Here we study the direct relationships between presence of LB nanotemplates and unique patterns of water molecules surrounding the protein, for four model proteins for which 3D structures are available, and where crystallization conditions for each protein are the same except the presence of LB nanotemplate.

Oxygen-bound Hell's gate globin I by classical versus LB nanotemplate method.

X-ray atomic structure of recombinant Hell's gate globin I (HGbI) from Methylacidophilum infernorum was calculated from the X-ray diffraction data of two different types of crystals: obtained by classical hanging drop and by LB nanotemplate method under the same crystallization conditions. After the accurate comparison of crystallographic parameters and electron density maps of two structures they appears to be quite similar, while the quality of the crystals grown by LB nanotemplate method was higher then of those grown by classical method. Indeed, the resolution of the LB crystal structure was 1.

Matrices for Sensors from Inorganic, Organic, and Biological Nanocomposites.

Matrices and sensors resulting from inorganic, organic and biological nanocomposites are presented in this overview. The term nanocomposite designates a solid combination of a matrix and of nanodimensional phases differing in properties from the matrix due to dissimilarities in structure and chemistry. The nanoocomposites chosen for a wide variety of health and environment sensors consist of Anodic Porous Allumina and P450scc, Carbon nanotubes and Conductive Polymers, Langmuir Blodgett Films of Lipases, Laccases, Cytochromes and Rhodopsins, Three-dimensional Nanoporous Materials and Nucleic Acid Programmable Protein Arrays.

In situ study of nanotemplate-induced growth of lysozyme microcrystals by submicrometer GISAXS.

Ultrasmall lysozyme microcrystals are grown by classical hanging-drop vapor diffusion and by its modification using a homologous protein thin-film template displaying long-range order. The nucleation and growth mechanisms of lysozyme microcrystals are studied at the thin lysozyme film surface using a new in situ µGISAXS (microbeam grazing-incidence small-angle X-ray scattering) technique recently developed at the microfocus beamline of the ESRF in Grenoble, France. New insight on the nucleation and crystallization processes appear to emerge.

In situ muGISAXS: II. Thaumatin crystal growth kinetic.

The formation of thaumatin crystals by Langmuir-Blodgett (LB) film nanotemplates was studied by the hanging-drop technique in a flow-through cell by synchrotron radiation micrograzing-incidence small-angle x-ray scattering. The kinetics of crystallization was measured directly on the interface of the LB film crystallization nanotemplate. The evolution of the micrograzing-incidence small-angle x-ray scattering patterns suggests that the increase in intensity in the Yoneda region is due to protein incorporation into the LB film.

In situ muGISAXS: I. Experimental setup for submicron study of protein nucleation and growth.

In this study, we used microbeam grazing-incidence small-angle x-ray scattering (muGISAXS) to investigate in situ protein nucleation and crystal growth assisted by a protein nanotemplate, and introduced certain innovations to improve the method. Our aim was to understand the protein nanotemplate method in detail, as this method has been shown to be capable of accelerating and increasing crystal size and quality, as well as inducing crystallization of proteins that are not crystallizable by classical methods. The nanotemplate experimental setup was used for drops containing growing protein crystals at different stages of nucleation and growth.

Domain organization and properties of LB lysozyme crystals down to submicron size.

New topographic details appeared evident in protein crystal buffered with glycerol solution native on mica by atomic force microscopy and after laser irradiation on glass by light microscopy. This observation indicates the existence of distinct domains in the 3D crystal organisation that are quite different in size and number between the lysozyme crystals grown by Langmuir-Blodgett (LB) nanotemplate with respect to traditional hanging-drop vapour diffusion. Nanodiffraction by highly focused synchrotron radiation of laser cut submicron crystals confirmed the atomic structure of all residues of LB lysozyme crystals as being the most resistant to radiation damage.