Nanoworld Institute

Genoa, Italy

Nanoworld Institute

Genoa, Italy

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Pechkova E.,Nanoworld Institute | Gebhardt R.,European Synchrotron Radiation Facility | Riekel C.,European Synchrotron Radiation Facility | Nicolini C.,Nanoworld Institute
Biophysical Journal | Year: 2010

In this study, we used microbeam grazing-incidence small-angle x-ray scattering (mGISAXS) 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. Two model proteins, lysozyme and thaumatin, were used under unique flow conditions to differentially probe protein crystal nucleation and growth. © 2010 by the Biophysical Society.

Gebhardt R.,European Synchrotron Radiation Facility | Gebhardt R.,TU Munich | Pechkova E.,Nanoworld Institute | Riekel C.,European Synchrotron Radiation Facility | Nicolini C.,Nanoworld Institute
Biophysical Journal | Year: 2010

The formation of thaumatin crystals by Langmuir-Blodgett (LB) film nanotemplates was studied by the hangingdrop 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 micro-grazing-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. The intensity variation suggests several steps, which were modeled by system dynamics based on first-order differential equations. The kinetic data can be described by two processes that take place on the LB film, a first, fast, process, attributed to the crystal growth and its detachment from the LB film, and a second, slower process, attributed to an unordered association and conversion of protein on the LB film. © 2010 by the Biophysical Society.

Pechkova E.,Nanoworld Institute | Nicolini C.,Fondazione ELBA
Journal of Synchrotron Radiation | Year: 2011

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. © 2011 International Union of Crystallography.

Bragazzi N.L.,University of Genoa | Bragazzi N.L.,Nanoworld Institute | Pechkova E.,University of Genoa | Pechkova E.,Nanoworld Institute | And 3 more authors.
Advances in Protein Chemistry and Structural Biology | Year: 2014

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. Nanobiotechnologies play a major role in this ambitious goal. In this review, we will focus on the bioinformatics, nanogenomics, and nanoproteomics aspects of contemporary nanodentistry, emphasizing the urgent need for an integrated proteogenomics approach and addressing its clinical and translational implications and new future perspectives and scenarios. © 2014 Elsevier Inc.

Spera R.,University of Genoa | Correia T.T.B.,University of Genoa | Nicolini C.,Nanoworld Institute | Nicolini C.,University of Genoa | Nicolini C.,Arizona State University
Sensors and Actuators, B: Chemical | Year: 2013

We present our preliminary results about the implementation of a new nanogravimetric biosensor realized combining Quartz Crystal Microbalance with Dissipation Monitoring Nanogravimetry and an innovative protein cell-free expression system named Nucleic Acid Programmable Protein Arrays (NAPPA) that allowed us to immobilize on the quartz surface, as sensing molecule, the proteins starting from its plasmid. We herby present results about the characterization of the nanogravimetric biosensor, its sensitivity, and its selectivity. Moreover we demonstrated how the simultaneous measurements of changes in resonance frequency and in energy dissipation were fundamental to correctly interpret the behavior of complex system such as NAPPA. © 2013 Elsevier B.V.

Nicolini C.,Nanoworld Institute | Nicolini C.,Arizona State University | Nicolini C.,Russian Academy of Sciences | Bruzzese D.,Nanoworld Institute | And 4 more authors.
Journal of Cellular Biochemistry | Year: 2013

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. The resulting expression plasmid, pET-28b, was under a strong T7/Lac promoter induced by IPTG (isopropyl-β-d-thiogalactoipyranoside). We obtained purification by fast protein liquid chromatography (FPLC) method. We recorded the variation of the current of a solution containing purified Laccase with increasing Syringaldazine (SGZ) concentration using a potentiometer as proof of principle, showing its compatibility with the development of a new enzymatic biosensor for medical purposes, as described in Part II. J. Cell. Biochem. 114: 599-605, 2013. © 2012 Wiley Periodicals, Inc.

Nicolini C.,Nanoworld Institute
Bioengineered | Year: 2013

This paper investigates the application of anodic porous alumina as an advancement on chip laboratory for gene expressions. The surface was prepared by a suitable electrolytic process to obtain a regular distribution of deep micrometric holes and printed bypen robot tips under standard conditions. The gene expression within the Nucleic Acid Programmable Protein Array (NAPPA) is realized in a confined environment of 16 spots, containing circular DNA plasmids expressed using rabbit reticulocyte lysate. Authors demonstrated the usefulness of APA in withholding the protein expression by detecting with a CCD microscope the photoluminescence signal emitted from the complex secondary antibody anchored to Cy3 and confined in the pores. Friction experiments proved the mechanical resistance under external stresses by the robot tip pens printing. So far, no attempts have been made to directly compare APA with any other surface/substrate; the rationale for pursuing APA as a potential surface coating is that it provides advantages over the simple functionalization of a glass slide, overcoming concerns about printing and its ability to generate viable arrays.

Nicolini C.,Nanoworld Institute
Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology | Year: 2010

This review presents the status of technological developments of nanogenomics and its applications to medicine. Even if particular emphasis is placed on what has been accomplished in our laboratory in the last few years in the area of genes microarrays, significant reference to the recent activity of numerous other groups can be found in Refs 1,2. © 2009 John Wiley & Sons, Inc.

Bragazzi N.L.,Nanoworld Institute | Sivozhelezov V.,Nanoworld Institute | Nicolini C.,Nanoworld Institute
Journal of Proteomics and Bioinformatics | Year: 2011

DNA microarrays are one of the most promising methods for molecular genomics, but this technique is often associated with experimental complications and difficulties in the analysis. Moreover, the greatest part of genes displayed on an array is often not directly involved in the cellular process being studied. Recently, we proposed a data mining algorithm, based on the identification of genes involved in a given process, the calculation of interactions among them and their ranking according to number of interactions. Genes in the highest cluster are defined as "leader genes". These findings may lead to an ad hoc and therefore more significant experimentation. However, at present this complex process is performed manually. In this work, we present the general architecture of LeaderGene, an automated tool for ab-initio molecular genomics. Three different and independent parts: (1) Identification of gene list; (2) Calculation of weighted number of links; (3) Genes clustering. Initial inputs are provided by user; then, output of part 1 and part 2, respectively, become inputs of parts 2 and 3. The development of an user-friendly software capable to automatically compute leader genes in a given cellular system will allow further progresses in this field of molecular genomics. © 2011 Bragazzi NL, et al.

Pechkova E.,Nanoworld Institute | Nicolini C.,Nanoworld Institute
Anticancer Research | Year: 2010

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. Crystals grown by LB nanotemplate still diffracted at good resolution after several steps of X-ray 'burning', while the classical crystals decayed very quickly at the same exposure.

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