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Albertazzi L.,CNR Institute of Neuroscience | Albertazzi L.,Center for Nanotechnology Innovation | Brondi M.,CNR Institute of Neuroscience | Brondi M.,Center for Nanotechnology Innovation | And 9 more authors.
PLoS ONE | Year: 2011

Background: The development of fluorescent proteins and synthetic molecules whose fluorescence properties are controlled by the environment makes it possible to monitor physiological and pathological events in living systems with minimal perturbation. A large number of small organic dyes are available and routinely used to measure biologically relevant parameters. Unfortunately their application is hindered by a number of limitations stemming from the use of these small molecules in the biological environment. Principal Findings: We present a novel dendrimer-based architecture leading to multifunctional sensing elements that can overcome many of these problems. Applications in vitro, in living cells and in vivo are reported. In particular, we image for the first time extracellular pH in the brain in a mouse epilepsy model. Conclusion: We believe that the proposed architecture can represent a useful and novel tool in fluorescence imaging that can be widely applied in conjunction with a broad range of sensing dyes and experimental setups. © 2011 Albertazzi et al.


De Vivo L.,CNR Institute of Neuroscience | Landi S.,CNR Institute of Neuroscience | Landi S.,Normal School of Pisa | Panniello M.,Normal School of Pisa | And 11 more authors.
Nature Communications | Year: 2013

Brain cells are immersed in a complex structure forming the extracellular matrix. The composition of the matrix gradually matures during postnatal development, as the brain circuitry reaches its adult form. The fully developed extracellular environment stabilizes neuronal connectivity and decreases cortical plasticity as highlighted by the demonstration that treatments degrading the matrix are able to restore synaptic plasticity in the adult brain. The mechanisms through which the matrix inhibits cortical plasticity are not fully clarified. Here we show that a prominent component of the matrix, chondroitin sulfate proteoglycans (CSPGs), restrains morphological changes of dendritic spines in the visual cortex of adult mice. By means of in vivo and in vitro two-photon imaging and electrophysiology, we find that after enzymatic digestion of CSPGs, cortical spines become more motile and express a larger degree of structural and functional plasticity. © 2013 Macmillan Publishers Limited.


Pavan G.M.,University of Applied Sciences and Arts Southern Switzerland | Albertazzi L.,CNR Institute of Neuroscience | Albertazzi L.,Center for Nanotechnology Innovation | Danani A.,University of Applied Sciences and Arts Southern Switzerland
Journal of Physical Chemistry B | Year: 2010

This paper reports a molecular dynamic study to explore the diverse behavior of different generations of poly(amidoamine) (PAMAM) dendrimers in binding siRNA. Our models show good accordance with experimental measurements. Simulations demonstrate that the molecular flexibility of PAMAMs plays a crucial role in the binding event, which is controlled by the modulation between enthalpy and entropy of binding. Importantly, the ability of dendrimers to adapt to siRNA is strongly dependent on the generation and on the pH due to backfolding. While G4 demonstrates good adaptability to siRNA, G6 behaves like a rigid sphere with a consistent loss in the binding affinity. G5 shows a hybrid behavior, maintaining rigid and flexible aspects, with a strong dependence of its properties on the pH. To define the "best binder", the mere energetic definition of binding affinity appears to be no longer effective and a novel concept of "efficiency" should be considered, being the balance between enthalpy and entropy of binding indivisible from the structural flexibility. With this aim, we propose an original criterion to define and rank the ability of these molecules to adapt their structure to bind a charged target. © 2010 American Chemical Society.


Dal Maschio M.,Italian Institute of Technology | Ghezzi D.,Italian Institute of Technology | Bony G.,Italian Institute of Technology | Alabastri A.,Italian Institute of Technology | And 9 more authors.
Nature Communications | Year: 2012

In utero electroporation is a powerful tool to transfect and manipulate neural-precursor cells of the rodent parietal cortex and their progeny in vivo. Although this technique can potentially target numerous brain areas, reliability of transfection in some brain regions is low or physical access is limited. Here we present a new in utero electroporation configuration based on the use of three electrodes, the relative position and polarities of which can be adjusted. The technique allows easy access and exceedingly reliable monolateral or bilateral transfection at brain locations that could only be sporadically targeted before. By improvement in the efficiency of the electrical field distribution, demonstrated here by a mathematical simulation, the multi-electrode configuration also extends the developmental timeframe for reliable in utero electroporation, allowing for the first time specific transfection of Purkinje cells in the rat cerebellum. © 2012 Macmillan Publishers Limited. All rights reserved.


Albertazzi L.,TU Eindhoven | Storti B.,Scuola Normale Superiore and Instituto Nanoscienze CNR and emailIITatNEST email | Brondi M.,Scuola Normale Superiore and Instituto Nanoscienze CNR and emailIITatNEST email | Sato S.S.,Scuola Normale Superiore and Instituto Nanoscienze CNR and emailIITatNEST email | And 3 more authors.
Journal of Visualized Experiments | Year: 2013

The development of fluorescent indicators represented a revolution for life sciences. Genetically encoded and synthetic fluorophores with sensing abilities allowed the visualization of biologically relevant species with high spatial and temporal resolution. Synthetic dyes are of particular interest thanks to their high tunability and the wide range of measureable analytes. However, these molecules suffer several limitations related to small molecule behavior (poor solubility, difficulties in targeting, often no ratiometric imaging allowed). In this work we introduce the development of dendrimer-based sensors and present a procedure for pH measurement in vitro, in living cells and in vivo. We choose dendrimers as ideal platform for our sensors for their many desirable properties (monodispersity, tunable properties, multivalency) that made them a widely used scaffold for several biomedical devices. The conjugation of fluorescent pH indicators to the dendrimer scaffold led to an enhancement of their sensing performances. In particular dendrimers exhibit reduced cell leakage, improved intracellular targeting and allow ratiometric measurements. These novel sensors were successfully employed to measure pH in living HeLa cells and in vivo in mouse brain.


Grasso G.,University of Catania | Salomone F.,University of Catania | Salomone F.,Center for Nanotechnology Innovation | Tundo G.R.,University of Rome Tor Vergata | And 11 more authors.
Journal of Inorganic Biochemistry | Year: 2012

Insulin degradation is a finely tuned process that plays a major role in controlling insulin action and most evidence supports IDE (insulin-degrading enzyme) as the primary degradative agent. However, the biomolecular mechanisms involved in the interaction between IDE and its substrates are often obscure, rendering the specific enzyme activity quite difficult to target. On the other hand, biometals, such as copper, aluminum and zinc, have an important role in pathological conditions such as Alzheimer's disease or diabetes mellitus. The metabolic disorders connected with the latter lead to some metallostasis alterations in the human body and many studies point at a high level of interdependence between diabetes and several cations. We have previously reported (Grasso et al., Chem. Eur. J. 17 (2011) 2752-2762) that IDE activity toward Aβ peptides can be modulated by metal ions. Here, we have investigated the effects of different metal ions on the IDE proteolytic activity toward insulin as well as a designed peptide comprising a portion of the insulin B chain (B20-30), which has a very low affinity for metal ions. The results obtained by different experimental techniques clearly show that IDE is irreversibly inhibited by copper(I) but is still able to process its substrates when it is bound to copper(II). © 2012 Elsevier Inc. All rights reserved.


Roddaro S.,CNR Institute of Neuroscience | Pescaglini A.,CNR Institute of Neuroscience | Ercolani D.,CNR Institute of Neuroscience | Sorba L.,CNR Institute of Neuroscience | And 3 more authors.
Nano Research | Year: 2011

The controlled tailoring of the energy distribution in an electron system opens the way to interesting new physics and device concepts, as demonstrated by research on metallic nanodevices during recent years. Here we investigate how Josephson coupling in a superconductor-InAs nanowire junction can be tuned by means of hot-electron injection and we show that a complete suppression of superconductive effects can be achieved using a power as low as 100 pW. Nanowires offer a novel design freedom as they allow axial and radial heterostructures to be defined as well as control over doping profiles, which can be crucial in the development of devices-such as nanorefrigerators-where precisely controlled and predictable energy barriers are mandatory. Our work provides estimates for unknown key thermal and electrical parameters, such as the electron-phonon coupling, in our InAs nanostructures. © 2010 Tsinghua University Press and Springer-Verlag Berlin Heidelberg.


Ferrari A.,CNR Institute of Neuroscience | Faraci P.,CNR Institute of Neuroscience | Cecchini M.,CNR Institute of Neuroscience | Cecchini M.,Center for Nanotechnology Innovation | And 2 more authors.
Biomaterials | Year: 2010

During development and regeneration of the mammalian nervous system, directional signals guide differentiating neurons toward their targets. Soluble neurotrophic molecules encode for preferential direction over long distances while the local topography is read by cells in a process requiring the establishment of focal adhesions. The mutual interaction between overlapping molecular and topographical signals introduces an additional level of control to this picture. The role of the substrate topography was demonstrated exploiting nanotechnologies to generate biomimetic scaffolds that control both the polarity of differentiating neurons and the alignment of their neurites. Here PC12 cells contacting nanogratings made of copolymer 2-norbornene ethylene (COC), were alternatively stimulated with Nerve Growth Factor, Forskolin, and 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic (8CPT-2Me-cAMP) or with a combination of them. Topographical guidance was differently modulated by the alternative stimulation protocols tested. Forskolin stimulation reduced the efficiency of neurite alignment to the nanogratings. This effect was linked to the inhibition of focal adhesion maturation. Modulation of neurite alignment and focal adhesion maturation upon Forskolin stimulation depended on the activation of the MEK/ERK signaling but were PkA independent. Altogether, our results demonstrate that topographical guidance in PC12 cells is modulated by the activation of alternative neuronal differentiation pathways. © 2009 Elsevier Ltd. All rights reserved.


PubMed | Center for Nanotechnology Innovation
Type: Journal Article | Journal: Journal of the American Chemical Society | Year: 2010

Reversibly photoswitchable (i.e., photochromic) fluorescent proteins open the way to a number of advanced bioimaging techniques applicable to living-cell studies such as sequential photolabeling of distinct cellular regions, innovative FRET schemes, or nanoscopy. Owing to the relevance of fluorescent proteins from Aequorea victoria (AFPs) for cell biology, a photochromic toolbox constituted by several AFPs is highly desirable. Here we introduce four new photochromic AFPs whose reversible photoswitching occurs between the native bright and a dark state at low illumination power, on account of a very efficient cis-trans photoisomerization. Most remarkably, the optical bistability of these AFPs derives from the single E222Q mutation in the primary sequence. Apparently, the E222Q substitution can restore the intrinsic photochromic behavior of the isolated chromophore. The significance of these mutants for high-resolution in vivo cell imaging is shown by means of photochromic FRET experiments.


PubMed | Center for Nanotechnology Innovation
Type: Journal Article | Journal: Journal of the American Chemical Society | Year: 2010

Polarity-dependent fluorescent probes are recently attracting interest for high-resolution cell imaging. Following a stepwise rational approach, we prepared and tested a toolbox of new coumarin derivatives tailored to in vivo imaging applications. Our compounds are characterized by a donor-(coumarin core)-acceptor molecular structure, where the electron donor is represented by alkylether or naphthyl groups, and the electron acceptor is represented by benzothiazene and cyano groups. Prior to synthesis, the substitution patterns were screened by computational methods to provide functional fluorescent derivatives easy to synthesize, and with excitation in the visible region of spectrum. We set up a robust synthetic procedure tunable on the substitution patterns to achieve. These coumarins possess excellent fluorescence quantum yields (up to 0.95), high molar extinction coefficients (up to 46,000 M(-1) cm(-1)), and large Stokes shifts. Furthermore, they display strong solvatochromism, being almost non-emissive in water and very fluorescent in less polar media (up to 780-fold enhancement in brightness). The solvatochromism of these compounds can be accounted for by a photophysical method encompassing two communicating excited states. When tested on cultured cells, the results showed that the developed coumarins were not harmful and their photophysical properties were unchanged compared to free solution. According to the determined solvatochromic properties, the coumarin fluorescence was detected only in the most lipophilic environments of the cell. The prepared compounds represent remarkable tools to investigate subtle biochemical processes in the cell environment after appropriate conjugation to biomolecules, and at the same time constitute the basis for further engineering of a new generation of biosensors.

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