Search

Abstract: Myotonic Dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults, characterized by a variety of multisystemic features and associated with cardiac anomalies. Among cardiac phenomena, conduction defects, ventricular arrhythmias, and dilated cardiomyopathy represent the main cause of sudden death in DM1 patients. Patient‐specific induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) represent a powerful in vitro model for molecular, biochemical, and physiological studies of disease in the target cells. Here, we used an Atomic Force Microscope (AFM) to measure the beating profiles of a large number of cells, organized in CM clusters (Beating Bodies, BBs), obtained from wild type (WT) and DM1 patients. We monitored the evolution over time of the frequency and intensity of the beating. We determined the variations between different BBs and over various areas of a single BB, caused by morphological and biomechanical variations. We exploited the AFM tip to apply a controlled force over the BBs, to carefully assess the biomechanical reaction of the different cell clusters over time, both in terms of beating frequency and intensity. Our measurements demonstrated differences between the WT and DM1 clusters highlighting, for the DM1 samples, an instability which was not observed in WT cells. We measured differences in the cellular response to the applied mechanical stimulus in terms of beating synchronicity over time and cell tenacity, which are in good agreement with the cellular behavior in vivo. Overall, the combination of hiPSC‐CMs with AFM characterization can become a new tool to study the collective movements of cell clusters in different conditions and can be extended to the characterization of the BB response to chemical and pharmacological stimuli. [ABSTRACT FROM AUTHOR]

The pressure for more energy demand is a strong driver for the construction of offshore oil and gas platforms. This also applies to the Adriatic Sea where, since I960, several gas fields have been commissioned. The continuous expansion of the gas industry may potentially have detrimental effects on marine communities. This may become particularly clear in such a peculiar semi-enclosed basin as the Adriatic Sea, which contains ecosystems that are subject to intensive anthropic pressures, such as unsustainable fishing and inputs of environmental pollutants. To date, still scant information is available on the environmental effects of gas platforms. Thus, to comply with the national Italian legislation and to support the development of protocols for the monitoring of offshore activities, multidisciplinary studies have been conducted and performed by CNR-ISMAR in cooperation with some private and public research institutes. A multidisciplinary approach based on chemical, biological and ecological investigations was performed to evaluate both the water column and the sediment quality near a gas platform. The overall results indicate moderate effects of platform-related environmental stress. [ABSTRACT FROM AUTHOR]

The pressure for more energy demand is a strong driver for the construction of offshore oil and gas platforms. This also applies to the Adriatic Sea where, since 1960, several gas fields have been commissioned. The continuous expansion of the gas industry may potentially have detrimental effects on marine communities. This may become particularly clear in such a peculiar semi-enclosed basin as the Adriatic Sea, which contains ecosystems that are subject to intensive anthropic pressures, such as unsustainable fishing and inputs of environmental pollutants. To date, still scant information is available on the environmental effects of gas platforms. Thus, to comply with the national Italian legislation and to support the development of protocols for the monitoring of offshore activities, multidisciplinary studies have been conducted and performed by CNR-ISMAR in cooperation with some private and public research institutes. A multidisciplinary approach based on chemical, biological and ecological investigations was performed to evaluate both the water column and the sediment quality near a gas platform. The overall results indicate moderate effects of platform-related environmental stress. [ABSTRACT FROM AUTHOR]

The study of the mechanical properties of biosystems and the relationship with their biochemical and structural functionality is an increasingly interesting subject of investigation. In recent years, in particular, the use of the atomic force microscopy provides the tools for understanding the molecular basis of the mechanical behaviour of the biosystems. The ageing of erythrocytes [red blood cells (RBCs)] constitutes a particularly interesting subject of study because of its fundamental role in triggering the cell turnover by promoting the removal of malfunctioning RBCs when specific ageing markers appear on their surface. Moreover, it is also interesting to study the role that the variation in the cells mechanical properties plays in the progress of the phenomenon. In this study, the ageing of RBCs, accelerated by depleting the cells of their ATP, has been investigated by two methods. The first is a recently developed nondestructive approach that correlates the roughness of the plasma membrane to the mechanical characteristics and the structural integrity of the cell membrane-skeleton. The second consists in directly measuring the nanomechanical properties by acquiring and analysing force curves on the cell membrane. The application of the two methods allowed to define, for the first time, the general scheme of alterations the cells experience during the ageing. In particular, a progressive decrease of the membrane roughness, correlated to a weakening of the membrane-skeleton support, and a complex pattern of changes in the nanomechanical properties, which drives the morphological variation and the occurrence of the specific ageing markers on the cells, have been revealed. Copyright © 2012 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

A review of the activity of scanning probe microscopy at our Institute is presented, going from instrumentation to software development of scanning tunnelling microscopy, atomic force microscopy and scanning near-field optical microscopy (SNOM). Some of the most important experiments in material science and biology performed by our group through the years with these SPM techniques will be presented. Finally, infrared applications by coupling a SNOM with a free electron laser will also be presented. [ABSTRACT FROM AUTHOR]

Introduction: The aim of this study is to investigate the nanocrystallization of steels caused by the transformation from the austenitic to the martensitic phase induced by a severe plastic deformation (SPD) treatment. In this framework, we applied an air blast shot peening treatment, which is a simple protocol widely used for industrial purposes. Methods: AISI 286 and AISI 316 specimens were peened for different times and polished using diamond pastes in order to remove corrugations higher than 1 μm. The characterization of the steel surfaces was performed by atomic force microscopy (AFM) operating in contact mode. Additional EDXD measurements were performed to confirm the phase transition. Results and Discussion: An AFM-based characterization at nanometric level of the steel surfaces is provided. When the peening exceeds a threshold time that, as expected, depends on the steel composition, a uniform nanostructuration is detected. It is well known that such rearrangement is associated to the growth of a martensitic phase. To date, AFM has been employed in this field only for few applications and to solve specific problems. On the other hand, our results demonstrate that this is a useful technique for the characterization of hardened surfaces, especially when non-destructive sample preparation treatments are required. Moreover, we show that AFM can be a useful tool also for in situ industrial diagnostics of metallic parts. [ABSTRACT FROM AUTHOR]

We present the implementation of a tapping-mode aperture scanning near-field optical microscope (Tapping-SNOM) to a Binder CB incubator (Istituto di Struttura della Materia, Rome, Italy). The microscope operates in the intermittent contact mode using a nonbent optical fibre allowing to reduce the perturbation exerted on the sample, while the incubator maintains a constant temperature, humidity and CO2 level. This instrument can maintain and analyse in a controlled environment different samples, both organic and nonorganic. In particular, the Tapping-SNOM can study different cell lines at nanometric resolution and in physiological buffer, following the evolution of the living cells almost indefinitely. We will present several examples of the capabilities of the tapping scanning near-field optical microscope in the study of different lines of living cells, showing corresponding topographical, optical or phase-lag images of the live samples, evidencing the excellent stability, versatility and resolution of the system. [ABSTRACT FROM AUTHOR]

We present the implementation of a tapping-mode aperture scanning near-field optical microscope (Tapping-SNOM) to a Binder CB incubator (Istituto di Struttura della Materia, Rome, Italy). The microscope operates in the intermittent contact mode using a nonbent optical fibre allowing to reduce the perturbation exerted on the sample, while the incubator maintains a constant temperature, humidity and CO2 level. This instrument can maintain and analyse in a controlled environment different samples, both organic and nonorganic. In particular, the Tapping-SNOM can study different cell lines at nanometric resolution and in physiological buffer, following the evolution of the living cells almost indefinitely. We will present several examples of the capabilities of the tapping scanning near-field optical microscope in the study of different lines of living cells, showing corresponding topographical, optical or phase-lag images of the live samples, evidencing the excellent stability, versatility and resolution of the system. [ABSTRACT FROM AUTHOR]

We investigated for the first time the haem stereochemistry in the nitrosylated derivative of two amphibian haemoglobins,Xenopus laevisandAmbystoma mexicanum, by means of X-ray absorption spectroscopy technique with the aim to explain the relationships between the active site structure and physiological function of these proteins, compared to that from humans. Our results show that while the Fe site local structure of human HbNO is modulated by an allosteric effector such as IHP shifting theT-Requilibrium towards theT-state, the Fe site local structure of amphibians HbNO is stabilized in a particularly tensedT-state also without IHP. [ABSTRACT FROM AUTHOR]

.Proteins of the transferrin (Tf) family have a role in metal transport in vertebrates and have been extensively studied. The results here reported provide, for the first time, a detailed systematic comparison of metal sites in Tf complexes involving several atoms in the whole protein and in two different types of Tfs. The high interest in the structural variations induced in a metalloprotein upon the uptake of different metals is related to the hypothesis of the metals' involvement in some neuropathologies. We propose a comparative study of the X-ray absorption spectra at the K-edge of iron, copper, zinc and nickel in serotransferrin and ovotransferrin. The experimental data are simulated using an algorithm of the full multiple scattering method. Our results show that: (1) the local structure of each site (N-terminal and C-terminal) is correlated to the ligation state of the other site; (2) the difference between the two proteins is related to site local structure and depends on the metal ion nature being greater in the case of copper and zinc with respect to iron and nickel ions; (3) X-ray spectroscopy is confirmed as a suitable technique able to discriminate between coordination models proposed by X-ray diffraction. [ABSTRACT FROM AUTHOR]

We used air operating atomic force microscopy (AFM) to study several morphological modifications of human erythrocytes, artificially produced by addition of exogenous agents including phospholipids, low ionic strength media and drugs. Most experiments were performed on unfixed samples to avoid treating red blood cells (RBCs) with chemical agents that can, in principle, induce morphological alteration. After detailed quantitative AFM characterization, the artificially produced abnormally shaped RBCs were compared with cells that occur with high incidence in blood pathologies. This morphological approach suggests a new strategy to describe and understand the biochemical and/or mechanical modifications responsible for the underlying pathologically induced changes and prove AFM to be a suitable tool to study erythrocyte deformation. [ABSTRACT FROM AUTHOR]

The Cu site structure of human serotransferrin and hen ovotransferrin using XANES spectroscopy has been investigated. Although the transferrin family proteins have been extensively studied, the results reported herein are the first concerning the structure of the metal site in C-terminal and N-terminal in the whole protein. Our structural data show that these proteins differ with regard to the independence of the two binding sites and the geometry of copper coordination, ranging from a poorly to a significantly distorted octahedron. [ABSTRACT FROM AUTHOR]

The Al site structure of serum transferrin and lactoferrin is investigated using X-ray absorption near edge structure (XANES) spectroscopy. Al K-edge spectra in the mono- and dialuminum forms of the proteins have been recorded for the first time. Our results show that the aluminium ion is hexa-coordinated in an octahedral-like symmetry and that the monoaluminum form, where only the C-terminal binding site is saturated, has an increased structural distortion around the metal site. [ABSTRACT FROM AUTHOR]

Abstract Spin resolved multiple scattering (MS) calculations of Fe K-edge X-ray Absorption Near Edge Structure (XANES) of myoglobin are reported for the first time. The observed differences of the Fe K-edge XANES spectra of sperm whale metmyoglobin under spin transition as a function of temperature have been studied. The method allows one to compute separately spin effects and local structural effects. The results show that spin effects are confined in the absorption rising edge in the range 7111-7130 eV, while purely structural effects are dominant in the range 7130-7170 eV. Symmetry changes of the Fe coordination sphere mainly related to its movement towards the heme plane, coupled to an increase of axial asymmetry, can explain the XANES changes observed above 7130 eV without an appreciable change of the Fe-N[sub p] distance. [ABSTRACT FROM AUTHOR]

Abstract. Atomic force microscopy (AFM) has been used to observe modifications induced by a 50-Hz 2-mT sinusoidal magnetic field on human B-lymphoblastoid cells (Raji). Cells exposed up to 64 h showed a drastic decrease in cell height during the first 13 h, reflecting the disappearance of microvilli, and a slower variation of the same parameter during the following 51 h together with a widening and loss of the cell's domed shape that corresponds to modifications at the cytoskeleton level. [ABSTRACT FROM AUTHOR]

Air operating atomic force microscopy has been used to study human B lymphoblastoid (Raji) cells exposed to a 50 Hz 2 mT sinusoidal magnetic field (MF), up to 64 h. A statistical analysis performed on mean height and membrane roughness of the cells showed a loss of microvilli and several modifications of the overall shape and membrane surface at increasing MF exposure. © 2001 American Institute of Physics. [ABSTRACT FROM AUTHOR]

The morphological changes of murine hematopoietic 32D Cl3-A cell line, in which apoptosis was induced by deprivation of interleukin-3 (IL-3) from the culture medium, were studied through air operating atomic force microscopy. The subtraction of IL-3 determines the appearing of blebs on plasma membrane and cell fragmentation in several residual bodies. Areas of strong contrast in the friction images, which are nearly absent in control and necrotic cells, were detected with percentage that peaks at 6 h of IL-3 deprivation. These are the results revealing that transient modification of the plasma membrane accompanies the first steps of apoptosis pathway. © 2001 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Erythrocytes (RBCs) constitute a very interesting class of cells both for their physiological function and for a variety of peculiarities. Due to their exceptionally strong relationship with the environment, the morphology and nanoscale characteristics of these cells can reveal their biochemical status and structural integrity. Among the possible subjects of investigations, the RBCs' ageing is of the utmost importance. This is a fundamental phenomenon that, in physiological conditions, triggers the cell turnover and ensures the blood homeostasis. With these premises, in recent years, we have presented an atomic force microscopy–based methodology to characterize the patterns of RBC ageing from the morphological point of view. In the present work, we used an ageing protocol more similar to the physiological conditions and we used differential scanning calorimetry and atomic force microscopy to probe the cross correlation between important structural and functional proteins. We also assessed the role played by fundamental structural and membrane proteins in the development of the most relevant morphological intermediates observed along the ageing. Furthermore, we coupled the morphological ageing patterns to the (bio)chemical alterations detected by Raman spectroscopy. This allowed identifying the chronology of the ageing morphologies and the metabolic pathways most involved in their development. As a whole, the present study provides the base to correlate specific molecular alterations to the development of structural anomalies, and these latter to the functional status of blood cells. We characterized the pattern of erythrocytes' ageing in starvation conditions through an experimental approach that combined atomic force microscopy (AFM), differential scanning calorimetry (DSC), and Raman spectro‐microscopy. Moreover, by using a DSC‐based procedure consisting in the warming and chilling of the samples at specific temperature capable to induce unfolding of specific cytoskeletal proteins, we were able to understand the role of selected molecular components in the development of structural anomalies and in the induction of specific ageing‐related morphologies. [ABSTRACT FROM AUTHOR]

The overall shape and membrane surface of human erythrocytes (RBCs) in the presence of nifedipine (a dihydropyridinic drug used in the clinical treatment of hypertension and angina pectoris) were imaged by contact-mode atomic force microscopy. Nifedipine induces in RBCs relevant morphological changes the extent of which increases as a function of drug concentration and incubation time. The modifications have been interpreted as mainly due to insertion of nifedipine into the outer layer of the RBC membrane. The potential effect of nifedipine as a hemoglobin denaturant has been ruled out by x-ray absorption near-edge structure and optical spectroscopies. © 2000 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Abstract. Vanadium oxide (V[sub 2]O[sub 5]) thin films, which are good candidates for the realization of gas detectors, have been grown on rough alumina substrates and characterized by atomic force microscopy (AFM). The films were prepared by radiofrequency reactive sputtering, with different concentrations of oxygen in the growth atmosphere; after deposition the samples were thermally treated in order to get the best sensitivity to different gases. Because of the structures already present on the rough alumina substrate, AFM showed large topographical variations (several hundred nanometers) for V[sub 2]O[sub 5] films grown on a rough alumina substrate. For both thermally treated and untreated samples, the roughness increases with increasing percentage of oxygen, but the sample with the best sensing properties (15% of oxygen) shows a characteristic minimum. Moreover, we observe that the thermal treatment also produces a general decrease in roughness, which is especially marked in samples with a low percentage of oxygen. The sensor has been tested with NO[sub 2], and the best sensitivity was obtained for an operating temperature around 300 Celsius. [ABSTRACT FROM AUTHOR]