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Andrea ALESSANDRINI

Professore Associato
Dipartimento di Scienze Fisiche, Informatiche e Matematiche sede ex-Fisica

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Pubblicazioni

2023 - Cytochalasin B Influences Cytoskeletal Organization and Osteogenic Potential of Human Wharton's Jelly Mesenchymal Stem Cells [Articolo su rivista]
Pampanella, L; Abruzzo, Pm; Tassinari, R; Alessandrini, A; Petrocelli, G; Ragazzini, G; Cavallini, C; Pizzuti, V; Collura, N; Canaider, S; Facchin, F; Ventura, C
abstract

Among perinatal stem cells of the umbilical cord, human Wharton's jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1-3 mu M) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers.

2023 - Dysregulation of NF–Y splicing drives metabolic rewiring and aggressiveness in colon cancer [Poster]
Belluti, S.; Mularoni, V.; Campani, Virginia; Rigillo, G.; Cuoghi, L.; Ronzio, M.; Miserocchi, G.; Dolfini, D.; Righi, Valeria; Alessandrini, A.; Zappavigna, V.; Imbriano, C.
abstract

NF-Y is an evolutionarily conserved transcription factor that binds specifically to the CCAAT elements of eukaryotic genes, most of which frequently deregulated in cancer. NF-YA, the regulatory subunit of the NF-Y complex, has two isoforms generated by alternative splicing, NF-YAl and NF-YAs, which differ in the transactivation domain. Transcriptomic data from The Cancer Genome Atlas (TCGA) database highlighted a significant increase in the expression of NF-YAs at the expense of NF-YAl in colorectal cancer (CRC), compared to healthy tissues. Despite this, high NF-YAl levels predict lower patients’ survival and distinguish the mesenchymal molecular subtype CMS4, which is characterized by the worst prognosis. Through the analysis of 3D cellular models, we demonstrated that altered expression of genes related to extracellular matrix and epithelial-mesenchymal transition sustains enhanced migratory and invasive behavior of NF-YAl-transduced cells. Moreover, the integration of metabolomics, bioenergetics and transcriptional analyses demonstrated a direct role for NFYAl in metabolic flexibility of cancer cells that adjust their metabolism in response to environmental changes to potentiate migration. The zebrafish xenograft model confirmed the metastatic potential triggered by NF-YAl in CRC cells. Altogether, our data highlight the transcriptional role of NF-YAl in CRC aggressiveness and suggest splice-switching strategies to hinder NF-YAl-induced metastatic dissemination.

2023 - The NF-YA Splicing Signature Controls Aggressiveness of Colon Cancer by Regulating Cell Metabolism and Different Types of Cell Migration [Relazione in Atti di Convegno]
Belluti, Silvia; Mularoni, Valentina; Rigillo, Giovanna; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-Y isoforms, NFYAs and NF-YAl, differentially control cell proliferation and differentiation. The analysis of patient’s transcriptome profiles from The Cancer Genome Atlas database highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA have a lower overall survival probability. We investigated the role of NF-YA in the metabolism of CRC cells, and the measurement of mitochondrial fuel usage in live cells shows that NF-YAl overexpression enhances the capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Moreover, we demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT, extracellular matrix and adhesion genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NFYAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patients’ stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities.

2022 - Acute cytotoxicity of mineral fibres observed by time-lapse video microscopy [Articolo su rivista]
Di Giuseppe, D.; Scarfi, S.; Alessandrini, A.; Bassi, A. M.; Mirata, S.; Almonti, V.; Ragazzini, G.; Mescola, A.; Filaferro, M.; Avallone, R.; Vitale, G.; Scognamiglio, V.; Gualtieri, A. F.
abstract

Inhalation of mineral fibres is associated with the onset of an inflammatory activity in the lungs and the pleura responsible for the development of fatal malignancies. It is known that cell damage is a necessary step for triggering the inflammatory response. However, the mechanisms by which mineral fibres exert cytotoxic activity are not fully understood. In this work, the kinetics of the early cytotoxicity mechanisms of three mineral fibres (i.e., chrysotile, crocidolite and fibrous erionite) classified as carcinogenic by the International Agency for Research on Cancer, was determined for the first time in a comparative manner using time-lapse video microscopy coupled with in vitro assays. All tests were performed using the THP-1 cell line, differentiated into M0 macrophages (M0-THP-1) and exposed for short times (8 h) to 25 μg/mL aliquots of chrysotile, crocidolite and fibrous erionite. The toxic action of fibrous erionite on M0-THP-1 cells is manifested since the early steps (2 h) of the experiment while the cytotoxicity of crocidolite and chrysotile gradually increases during the time span of the experiment. Chrysotile and crocidolite prompt cell death mainly via apoptosis, while erionite exposure is also probably associated to a necrotic-like effect. The potential mechanisms underlying these different toxicity behaviours are discussed in the light of the different morphological, and chemical-physical properties of the three fibres.

2022 - Alternative splicing of the transcription factor NF-Y promotes cell migration and invasion in colon cancer [Abstract in Atti di Convegno]
Rigillo, Giovanna; Belluti, Silvia; Campani, Virginia; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

The heterotrimeric transcription factor NF-Y directly controls the expression of genes involved in cellular pathways commonly altered in cancer cells, such as cell cycle, apoptosis and metabolism. Consistently, the binding site for NF-Y is highly enriched in the regulatory regions of genes overexpressed in tumors, and mRNA levels of NF-Y subunits are altered in cancer tissues and cells. In particular, the DNA binding subunit NF-YA is up-regulated in various tumors, among which gastric, lung, breast, ovarian, osteosarcoma and prostate cancers. Moreover, a switch between the two alternatively NF-YA spliced transcripts, NF-YAs and NF-YAl, occurs in tumor tissues compared to normal ones. Colorectal cancer (CRC) is the third most common malignancy worldwide. Four internationally approved consensus molecular subtypes (CMS) represent the best current description of CRC heterogeneity at the gene-expression level: the CMS1 group is characterized by the immune infiltration signature, CMS2 is the canonical epithelial subtype, CMS3 represents the metabolic group, and CMS4 is the mesenchymal one, associated with a worse prognosis and poor response to therapies compared to other subtypes. Here we show that increased levels of NF-YA characterize CRC versus healthy tissues. We identified a significant association between NF-YA isoforms and CRC subtypes: NF-YAs is up-regulated in all CMSs in opposition to NF-YAl, which is down-regulated in all subtypes with the exception of aggressive and metastatic CMS4 group. By using in vitro cell models, we confirmed that NF-YAs is the predominant isoform in CRC cell lines, while NF-YAl levels proportionally increase from epithelial to hybrid and mesenchymal cells. The modulation of NF-YA isoforms in CRC cells significantly affects cancer cell behavior by modulating differently, even oppositely, the transcription of genes associated to extracellular-matrix (ECM) and epithelial-to-mesenchymal transition (EMT). We described different modes of migration and invasion properties for NF-YAs and NF-YAl overexpressing cells by using 2D and 3D culture conditions, time-lapse imaging of CRC cells and intravascular distribution of NF-YAs/l transduced CRC cells in the embryonic zebrafish xenograft model. Altogether, our data highlight the direct role of the longer NF-YA isoform in CRC cell dissemination and suggest its possible use as biomarker for molecular stratification predictive of progressive disease in CRC patients.

2022 - Chemical characterization of non-psychoactive Cannabis sativa L. extracts, in vitro antiproliferative activity and induction of apoptosis in chronic myelogenous leukaemia cancer cells [Articolo su rivista]
Anceschi, L.; Codeluppi, A.; Brighenti, V.; Tassinari, R.; Taglioli, V.; Marchetti, L.; Roncati, L.; Alessandrini, A.; Corsi, L.; Pellati, F.
abstract

In this study, extracts from non-psychoactive Cannabis sativa L. varieties were characterized by means of ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) and their antiproliferative activity was assessed in vitro. The human chronic myelogenous leukaemia cell line K562 was chosen to investigate the mechanism of cell death. The effect on the cell cycle and cell death was analysed by flow cytometry. Proteins related to apoptosis were studied by western blotting. Mechanical properties of cells were assessed using the Micropipette Aspiration Technique (MAT). The results indicated that the cannabidiol (CBD)-rich extract inhibited cell proliferation of K562 cell line in a dose-dependent manner and induced apoptosis via caspase 3 and 7 activation. A significant decrease in the mitochondrial membrane potential was detected, together with the release of cytochrome c into the cytosol. The main apoptotic markers were not involved in the mechanism of cell death. The extract was also able to modify the mechanical properties of cells. Thus, this hemp extract and its pure component CBD deserve further investigation for a possible application against myeloproliferative diseases, also in association with other anticancer drugs.

2022 - Cytochalasin B Modulates Nanomechanical Patterning and Fate in Human Adipose-Derived Stem Cells [Articolo su rivista]
Bianconi, Eva; Tassinari, Riccardo; Alessandrini, Andrea; Ragazzini, Gregorio; Cavallini, Claudia; Abruzzo, Provvidenza Maria; Petrocelli, Giovannamaria; Pampanella, Luca; Casadei, Raffaella; Maioli, Margherita; Canaider, Silvia; Facchin, Federica; Ventura, Carlo
abstract

Cytoskeletal proteins provide architectural and signaling cues within cells. They are able to reorganize themselves in response to mechanical forces, converting the stimuli received into specific cellular responses. Thus, the cytoskeleton influences cell shape, proliferation, and even differentiation. In particular, the cytoskeleton affects the fate of mesenchymal stem cells (MSCs), which are highly attractive candidates for cell therapy approaches due to their capacity for self-renewal and multi-lineage differentiation. Cytochalasin B (CB), a cyto-permeable mycotoxin, is able to inhibit the formation of actin microfilaments, resulting in direct effects on cell biological properties. Here, we investigated for the first time the effects of different concentrations of CB (0.1–10 μM) on human adipose-derived stem cells (hASCs) both after 24 h (h) of CB treatment and 24 h after CB wash-out. CB influenced the metabolism, proliferation, and morphology of hASCs in a dose-dependent manner, in association with progressive disorganization of actin microfilaments. Furthermore, the removal of CB highlighted the ability of cells to restore their cytoskeletal organization. Finally, atomic force microscopy (AFM) revealed that cytoskeletal changes induced by CB modulated the viscoelastic properties of hASCs, influencing their stiffness and viscosity, thereby affecting adipogenic fate

2022 - The NF-YA splicing signature controls aggressiveness of colon cancer by regulating different modes of cell migration and cell metabolism. [Poster]
Belluti, Silvia; Rigillo, Giovanna; Mularoni, Valentina; Ronzio, Mirko; Miserocchi, Giacomo; Dolfini, Diletta; Mercatali, Laura; Alessandrini, Andrea; Imbriano, Carol
abstract

NF-Y is a transcription factor composed of NF-YA and NF-YB/NF-YC subunits. The two NF-YA isoforms, NF-YAs and NF-YAl, differentially control cell proliferation and differentiation. TCGA data highlight increased NF-YA expression, specifically NF-YAs, in colorectal cancer (CRC), the second most deadly cancer worldwide. Despite this, patients with high NF-YAl mRNA levels have a lower overall survival probability. We demonstrate that NF-YAl overexpression can generate a hybrid epithelial-mesenchymal transition (EMT) state in CRC cells by direct transcriptional regulation of key EMT genes. Consistently, NF-YAl enhances cell migration, both in 2D and 3D culture conditions, as highlighted by live imaging investigations. While collective migration characterizes NF-YAs-cells, fast single-cell and amoeboid-like migration marks NF-YAl cells. In agreement with these results, NF-YAl overexpression promotes cell dissemination in zebrafish xenografts. Since metabolic reprogramming is a hallmark of cancer and metastasis, we also investigated the role of NF-YAl in the metabolism of CRC cells. The measure of mitochondrial fuel usage in live cells showed that NF-YAl overexpression enhances the dependency and capacity for glutamine pathway, one of the key metabolic pathways involved in EMT and cell dissemination. Specifically, we identified NF-YAl as direct transcriptional regulator of GLS1 glutaminase and GLUL glutamine synthetase. Our observations imply that the two NF-YA variants can be potentially novel markers for CRC patient stratification. Higher NF-YAl expression can be a hallmark of cancer cell dissemination by affecting cell metabolism and cell migratory abilities.

2022 - The potential of AFM in studying the role of the nanoscale amphipathic nature of (lipo)-peptides interacting with lipid bilayers [Articolo su rivista]
Mescola, A.; Ragazzini, G.; Facci, P.; Alessandrini, A.
abstract

Antimicrobial peptides (AMPs) and lipopeptides (LPs) represent very promising molecules to fight resistant bacterial infections due to their broad-spectrum of activity, their first target, i.e. the bacterial membrane, and the rapid bactericidal action. For both types of molecules, the action mechanism starts from the membrane of the pathogen agents, producing a disorganization of their phase structure or the formation of pores of different size altering their permeability. This mechanism of action is based on physical interactions more than on a lock-and-key recognition event and it is difficult for the pathogens to rapidly develop an effective resistance. Very small differences in the sequence of both AMPs and LPs might lead to very different effects on the target membrane. Therefore, a correct understanding of their mechanism of action is required with the aim of developing new synthetic peptides, analogues of the natural ones, with specific and more powerful bactericidal activity. Atomic force microscopy (AFM), with its high resolution and the associated force spectroscopy resource, provides a valuable technique to investigate the reorganization of lipid bilayers exposed to antimicrobial or lipopeptides. Here, we present AFM results obtained by ours and other groups on the action of AMPs and LPs on supported lipid bilayers (SLBs) of different composition. We also consider data obtained by fluorescence microscopy to compare the AFM data with another technique which can be used on different lipid bilayer model systems such as SLBs and giant unilamellar vesicles. The outcomes here presented highlight the powerful of AFM-based techniques in detecting nanoscale peptide-membrane interactions and strengthen their use as an exceptional complementary tool to in vivo investigations. Indeed, the combination of these approaches can help decipher the mechanisms of action of different antimicrobials and lipopeptides at both the micro and nanoscale levels, and to design new and more efficient antimicrobial compounds.

2021 - A Fully Integrated Arduino-Based System for the Application of Stretching Stimuli to Living Cells and Their Time-Lapse Observation: A Do-It-Yourself Biology Approach [Articolo su rivista]
Ragazzini, G.; Guerzoni, J.; Mescola, A.; Di Rosa, D.; Corsi, L.; Alessandrini, A.
abstract

Mechanobiology has nowadays acquired the status of a topic of fundamental importance in a degree in Biological Sciences. It is inherently a multidisciplinary topic where biology, physics and engineering competences are required. A course in mechanobiology should include lab experiences where students can appreciate how mechanical stimuli from outside affect living cell behaviour. Here we describe all the steps to build a cell stretcher inside an on-stage cell incubator. This device allows exposing living cells to a periodic mechanical stimulus similar to what happens in physiological conditions such as, for example, in the vascular system or in the lungs. The reaction of the cells to the periodic mechanical stretching represents a prototype of a mechanobiological signal integrated by living cells. We also provide the theoretical and experimental aspects related to the calibration of the stretcher apparatus at a level accessible to researchers not used to dealing with topics like continuum mechanics and analysis of deformations. We tested our device by stretching cells of two different lines, U87-MG and Balb-3T3 cells, and we analysed and discussed the effect of the periodic stimulus on both cell reorientation and migration. We also discuss the basic aspects related to the quantitative analysis of the reorientation process and of cell migration. We think that the device we propose can be easily reproduced at low-cost within a project-oriented course in the fields of biology, biotechnology and medical engineering.

2021 - An overview on microfluidic systems for nucleic acids extraction from human raw samples [Articolo su rivista]
Obino, D.; Vassalli, M.; Franceschi, A.; Alessandrini, A.; Facci, P.; Viti, F.
abstract

Nucleic acid (NA) extraction is a basic step for genetic analysis, from scientific research to diagnostic and forensic applications. It aims at preparing samples for its application with biomolecular technologies such as isothermal and non‐isothermal amplification, hybridization, electrophoresis, Sanger sequencing and next‐generation sequencing. Multiple steps are involved in NA collection from raw samples, including cell separation from the rest of the specimen, cell lysis, NA isolation and release. Typically, this process needs molecular biology facilities, specialized instrumentation and labor‐intensive operations. Microfluidic devices have been developed to analyze NA samples with high efficacy and sensitivity. In this context, the integration within the chip of the sample preparation phase is crucial to leverage the promise of portable, fast, user-friendly and economic point‐of‐care solutions. This review presents an overview of existing lab‐on-a‐chip (LOC) solutions designed to provide automated NA extraction from human raw biological fluids, such as whole blood, excreta (urine and feces), saliva. It mainly focuses on LOC implementation aspects, aiming to describe a detailed panorama of strategies implemented for different human raw sample preparations.

2021 - Antibacterial Effect of Stainless Steel Surfaces Treated with a Nanotechnological Coating Approved for Food Contact [Articolo su rivista]
Di Cerbo, Alessandro; Mescola, Andrea; Rosace, Giuseppe; Stocchi, Roberta; Rossi, Giacomo; Alessandrini, Andrea; Preziuso, Silvia; Scarano, Antonio; Rea, Stefano; Loschi, Anna Rita; Sabia, Carla
abstract

Stainless steel, widely present in the food industry, is frequently exposed to bacterial colonization with possible consequences on consumers’ health. 288 stainless steel disks with different roughness (0.25, 0.5 and 1 μm) were challenged with four Gram-negative (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 1402, Yersinia enterocolitica ATCC 9610 and Pseudomonas aeruginosa ATCC 27588) and four Gram-positive bacteria (Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 14579 and Listeria monocytogenes NCTT 10888) and underwent three different sanitizing treatments (UVC, alcohol 70% v/v and Gold lotion). Moreover, the same procedure was carried out onto the same surfaces after a nanotechnological surface coating (nanoXHAM® D). A significant bactericidal effect was exerted by all of the sanitizing treatments against all bacterial strains regardless of roughness and surface coating. The nanoXHAM® D coating itself induced an overall bactericidal effect as well as in synergy with all sanitizing treatments regardless of roughness. Stainless steel surface roughness is poorly correlated with bacterial adhesion and only sanitizing treatments can exert significant bactericidal effects. Most of sanitizing treatments are toxic and corrosive causing the onset of crevices that are able to facilitate bacterial nesting and growth. This nanotechnological coating can reduce surface adhesion with consequent reduction of bacterial adhesion, nesting, and growth

2021 - Enhanced Osteogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells by a Hybrid Hydroxylapatite/Collagen Scaffold [Articolo su rivista]
Mazzoni, E.; Mazziotta, C.; Iaquinta, M. R.; Lanzillotti, C.; Fortini, F.; D'Agostino, A.; Trevisiol, L.; Nocini, R.; Barbanti-Brodano, G.; Mescola, A.; Alessandrini, A.; Tognon, M.; Martini, F.
abstract

Human bone marrow-derived mesenchymal stem cells (hBMSCs) and their derivative enhanced green fluorescent protein (eGFP)-hBMSCs were employed to evaluate an innovative hybrid scaffold composed of granular hydroxylapatite and collagen hemostat (Coll/HA). The cellular morphology/cytoskeleton organization and cell viability were investigated by immunohistochemistry (IHC) and AlamarBlue metabolic assay, respectively. The expression of osteopontin and osteocalcin proteins was analyzed by IHC and ELISA, whereas osteogenic genes were investigated by quantitative PCR (Q-PCR). Cell morphology of eGFP-hBMSCs was indistinguishable from that of parental hBMSCs. The cytoskeleton architecture of hBMSCs grown on the scaffold appeared to be well organized, whereas its integrity remained uninfluenced by the scaffold during the time course. Metabolic activity measured in hBMSCs grown on a biomaterial was increased during the experiments, up to day 21 (p < 0.05). The biomaterial induced the matrix mineralization in hBMSCs. The scaffold favored the expression of osteogenic proteins, such as osteocalcin and osteopontin. In hBMSC cultures, the scaffold induced up-regulation in specific genes that are involved in ossification process (BMP2/3, SPP1, SMAD3, and SP7), whereas they showed an up-regulation of MMP9 and MMP10, which play a central role during the skeletal development. hBMSCs were induced to chondrogenic differentiation through up-regulation of COL2A1 gene. Our experiments suggest that the innovative scaffold tested herein provides a good microenvironment for hBMSC adhesion, viability, and osteoinduction. hBMSCs are an excellent in vitro cellular model to assay scaffolds, which can be employed for bone repair and bone tissue engineering.

2021 - Green Fabrication of (6,5)Carbon Nanotube/Protein Transistor Endowed with Specific Recognition [Articolo su rivista]
Berto, M.; Di Giosia, M.; Giordani, M.; Sensi, M.; Valle, F.; Alessandrini, A.; Menozzi, C.; Cantelli, A.; Gazzadi, G. C.; Zerbetto, F.; Calvaresi, M.; Biscarini, F.; Bortolotti, C. A.
abstract

A general single-step approach is introduced for the green fabrication of hybrid biosensors from water dispersion. The resulting device integrates the semiconducting properties of a carbon nanotube (CNT) and the functionality of a protein. In the initial aqueous phase, the protein (viz., lysozyme [LZ]) disperses the (6,5)CNT. Drop-casting of the dispersion on a test pattern (a silicon wafer with interdigitated Au source and drain electrodes) yields a fully operating, robust, electrolyte-gated transistor (EGT) in one step. The EGT response to biorecognition is then assessed using the LZ inhibitor N-acetyl glucosamine trisaccharide. Analysis of the output signal allows one to extract a protein-substrate binding constant in line with values reported for the free (without CNT) system. The methodology is robust, easy to optimize, redirectable toward different targets and sets the grounds for a new class of CNT-protein biosensors that overcome many limitations of the technology of fabrication of CNT biosensors.

2021 - IS PREGNENOLONE SULFATE AN ENDOGENOUS MODULATOR OF NICOTINIC ACH RECEPTOR? [Abstract in Atti di Convegno]
Puja, Giulia; Ravazzini, Federica; Balleza, Daniel; Alessandrini, Andrea
abstract

Pregnenolone sulfate (PregS) is an important endogenous modulator of brain activity. PregS reduces the inhibitory activity of GABAA receptors, increases excitatory neurotransmission potentiating NMDA receptors function, trafficking and glutamate release. These latter actions are mediated by PregS at the level of sigma I receptor, calcium and TRP channels [1]. The peleiotropic mechanism of action of PregS could explain some of the pharmacological effects of the steroid: in preclinical studies PregS is neuroprotective, it potentiates synaptic LTP and improve spatial cognitive performance in rats eventhough the pro-mnestic effect of PregS likely depends on the mode of administration and behavioral model being tested [2]. Because cholinergic transmission is fundamental for cognitive functions such as learning and memory we studied the effect of PregS on the activity of nicotinic Ach receptors (nAchR). Using the patch-clamp technique in the whole-cell configuration we tested PregS on the currents mediated by nAChRs expressed in SH-SY5Y cells and in neurons grown in primary cultures. In SH-SY5Y application of PregS reduced NIC (10 μM) - evoked currents with an IC50 in the low micromolar range (14+9 μM) and a maximal effect of 58+10 %. To investigate the mechanism of PregS antagonism we applied the same concentration (10 μM) of the neurosteroid together with different concentrations of NIC (from 5 to 100 μM). PregS effect was not dependent on agonist concentration suggesting a non competitive mechanism of action. The analysis of the dose-response curves of PregS effect at 10 and 100 μM NIC evidentiates that potency and efficacy of the steroid is unchanged. PregS reduces nAchR-mediated currents by increasing receptor desensitization and this effect is more pronounced at high agonist concentrations. Experiments in primary cultures of cortical and cerebellar neurons have confirmed the antagonistic effect of PregS on NIC-evoked current. The inhibitory effect of PregS of nAchR- mediated current cannot support its activity as cognitivie enhancer. To find a rationale it has to be taken into account an additional complexity resulting from the activity of PregS on a neuronal network: it is possible that the reduction of cholinergic activity be limited to specific inhibitory circuits and that the overall effect will be an improved excitatory neurotransmission. It is also conceivable that PregS effect could be served as a compensatory mechanism

2020 - Antibacterial effect of aluminum surfaces untreated and treated with a special anodizing based on titanium oxide approved for food contact [Articolo su rivista]
Di Cerbo, Alessandro; Mescola, Andrea; Iseppi, Ramona; Canton, Roberto; Rossi, Giacomo; Stocchi, Roberta; Rita Loschi, Anna; Alessandrini, Andrea; Rea, Stefano; Sabia, Carla
abstract

One of the main concerns of the food industry is microbial adhesion to food contact surfaces and consequent contamination. We evaluated the potential bacteriostatic/bactericidal ecacy of aluminum surfaces with dierent large-scale roughness (0.25, 0.5 and 1 m) before and after the surface treatment with a special anodizing based on titanium oxide nanotechnology (DURALTI®) and after 3 dierent sanitizing treatments, e.g., UV, alcohol and a natural product named Gold lotion. Four Gram-negative (Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 1402, Yersinia enterocolitica ATCC 9610 and Pseudomonas aeruginosa ATCC 27588) and four Gram-positive (Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 14579 and Listeria monocytogenes NCTT 10888) bacteria were screened. As far as concerns aluminum surfaces without nanotechnology surface treatment, an overall bacteriostatic eect was observed for all strains with respect to the initial inoculum that was 106 CFU/mL. Conversely, an overall bactericidal eect was observed both for Gram-negative and -positive bacteria on DURALTI®-treated aluminum disks, regardless of roughness and sanitizing treatment. These results are innovative in terms of the great potential of the antibacterial activity of nanotechnologically treated food contact surfaces and their combination with some sanitizing agents that might be exploited in the food industry

2020 - Daptomycin Strongly Affects the Phase Behavior of Model Lipid Bilayers [Articolo su rivista]
Mescola, A.; Ragazzini, G.; Alessandrini, A.
abstract

Daptomycin (DAP) is a calcium-dependent cyclic lipopeptide with great affinity for negatively charged phospholipids bearing the phosphatidylglycerol (PG) headgroup and has been used since 2003 as a last resort antibiotic in the treatment of severe infections caused by Gram-positive bacteria. The first step of its mechanism of action involves the interaction with the bacterial membrane, which not only represents a physical barrier but also accommodates transmembrane proteins, such as receptors, transporters, and enzymes, whose activity is crucial for the survival of bacteria. This results in a less efficient development of resistance strategies by pathogens compared to common antibiotics that activate or inhibit biochemical pathways connected to specific target proteins. Although already on the market, the molecular mechanism of action of DAP is still a controversial subject of investigation and it is most likely the result of a combination of distinct effects. Understanding how DAP targets the membrane of pathogens could be of great help in finding its analogues that could better avoid the development of resistance. Here, exploiting fluorescence microscopy and atomic force microscopy (AFM), we demonstrated that DAP affects the thermodynamic behavior of lipid mixtures containing PG moieties. Regardless of whether the PG lipids are in the liquid or solid phase, DAP preferably interacts with this headgroup and is able to penetrate more deeply into the lipid bilayer in the regions where this headgroup is present. In particular, considering the results of an AFM/spectroscopy investigation, DAP appears to produce a stiffening effect of the domains where PG lipids are mainly in the fluid phase, whereas it causes fluidification of the domains where PG lipids are in the solid phase.

2020 - Model lipid systems and their use to evaluate the phase state of biomembranes, their mechanical properties and the effect of non-conventional antibiotics: the case of daptomycin [Articolo su rivista]
Balleza, D.; Mescola, A.; Alessandrini, A.
abstract

The lipid bilayer is the basis of the structure and function of the cell membrane. The study of the molecular phenomena that affect biological membranes has a great impact on the understanding of cellular physiology. To understand these phenomena, it has become increasingly necessary to develop simple synthetic models that allow the most basic details of such processes to be reproduced. In this short communication, we took advantage of the properties of two well-established lipid model systems, GUVs and SLBs, with compositions mimicking the cell membrane present in mammals and bacteria, to study the thermotropic phase behavior of lipids as well as the effect of daptomycin, a cyclic lipopeptide used as an antibiotic. The study of mechanical and thermodynamical properties of these model systems could contribute to establish a theoretical framework to develop more efficient strategies for biological control.

2019 - A new approach for the separation, characterization and testing of potential prionoid protein aggregates through hollow-fiber flow field-flow fractionation and multi-angle light scattering [Articolo su rivista]
Marassi, Valentina; Beretti, Francesca; Roda, Barbara; Alessandrini, Andrea; Facci, Paolo; Maraldi, Tullia; Zattoni, Andrea; Reschiglian, Pierluigi; Portolani, Marinella
abstract

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

2019 - Complex Phase Behavior of GUVs Containing Different Sphingomyelins [Articolo su rivista]
Balleza, D; Mescola, A; Medina, N; Ragazzini, Gregorio; Pieruccini, M; Facci, P; Alessandrini, A
abstract

Understanding the lateral organization of biological membranes plays a key role on the road to fully appreciate the physiological functions of this fundamental barrier between the inside and outside regions of a cell. Ternary lipid bilayers composed of a high and a low melting temperature lipid and cholesterol represent a model system that mimics some of the important thermodynamical features of much more complex lipid mixtures such as those found in mammal membranes. The phase diagram of these ternary mixtures can be studied exploiting fluorescence microscopy in giant unilamellar vesicles, and it is typically expected to give rise, for specific combinations of composition and temperature, to regions of two-phase coexistence and a region with three-phase coexistence, namely, the liquid-ordered, liquid-disordered, and solid phases. Whereas the observation of two-phase coexistence is routinely possible using fluorescence microscopy, the three-phase region is more elusive to study. In this article, we show that particular lipid mixtures containing diphytanoyl-phosphatidylcholine and cholesterol plus different types of sphingomyelin (SM) are prone to produce bilayer regions with more than two levels of fluorescence intensity. We found that these intensity levels occur at low temperature and are linked to the copresence of long and asymmetric chains in SMs and diphytanoyl-phosphatidylcholine in the lipid mixtures. We discuss the possible interpretations for this observation in terms of bilayer phase organization in the presence of sphingolipids. Additionally, we also show that in some cases, liposomes in the three-phase coexistence state exhibit extreme sensitivity to lateral tension. We hypothesize that the appearance of the different phases is related to the asymmetric structure of SMs and to interdigitation effects.

2019 - Composti antitumorali [Brevetto]
Corsi, Lorenzo; Alessandrini, Andrea; Ettari, Roberta; Bennaud, Christelle
abstract

L’invenzione riguarda l’uso di derivati derivato dell’1-(4-amino-3,5-dimetilfenil)-3,5-diidro-7,8-etilenediossi-4h-2,3-benzodiazepin-4-one come agenti antitumorali.

2019 - Fabrication of a low-cost on-stage cell incubator with full automation [Articolo su rivista]
Regazzini, Gregorio; Mercola, Andrea; Corsi, Lorenzo; Alessandrini, Andrea
abstract

In recent years the possibility of observing by microscopy the dynamic activity of living cells has been largely pursued. We have developed a low-cost (~ 260 euros) on-stage cell incubator for inverted optical microscopes. This device allows to keep cells in good conditions for their survival and proliferation. The device is based on the use of the Arduino microprocessor interfaced with LabView. It can be connected to a computer via USB port allowing to monitor and register all the useful parameters of the measurements: temperature, CO2 concentration and relative humidity. It consists of a closed metallic and plastic (PMMA) chassis which provides optical transparency to the petri dish in order to use interference contrast imaging techniques. The system exploits also a second Arduino microprocessor to perform autofocus of the images and to automatically acquire images at defined time intervals. Cell biology laboratories could easily construct this device to allow also students to follow dynamic processes of living cells and to practice with the DIY (Do-It-Yourself) approach to biology. At the same time, students could become familiar with the use of low-cost microprocessors like Arduino.

2019 - Glutamate Receptors and Glioblastoma Multiforme: An Old "Route" for New Perspectives [Articolo su rivista]
Corsi, L; Mescola, A; Alessandrini, A
abstract

Glioblastoma multiforme (GBM) is the most aggressive malignant tumor of the central nervous system, with poor survival in both treated and untreated patients. Recent studies began to explain the molecular pathway, comprising the dynamic structural and mechanical changes involved in GBM. In this context, some studies showed that the human glioblastoma cells release high levels of glutamate, which regulates the proliferation and survival of neuronal progenitor cells. Considering that cancer cells possess properties in common with neural progenitor cells, it is likely that the functions of glutamate receptors may affect the growth of cancer cells and, therefore, open the road to new and more targeted therapies.

2019 - Magainin-H2 effects on the permeabilization and mechanical properties of giant unilamellar vesicles [Articolo su rivista]
Mescola, A.; Marin-Medina, N.; Ragazzini, G.; Accolla, M.; Alessandrini, A.
abstract

Among the potential novel therapeutics to treat bacterial infections, antimicrobial peptides (AMPs) are a very promising substitute due to their broad-spectrum activity and rapid bactericidal action. AMPs strongly interact with the bacterial membrane, and the need to have a correct understanding of the interaction between AMPs and lipid bilayers at a molecular level prompted a wealth of experimental and theoretical studies exploiting a variety of AMPs. Here, we studied the effects of magainin H2 (Mag H2), an analog of the well-known magainin 2 (wt Mag 2) AMP endowed with a higher degree of hydrophobicity, on giant unilamellar vesicles (GUVs) concentrating on its permeabilization activity and the effect on the lipid bilayer mechanical properties. We demonstrated that the increased hydrophobicity of Mag H2 affects its selectivity conferring a strong permeabilization activity also on zwitterionic lipid bilayers. Moreover, when lipid mixtures including PG lipids are considered, PG has a protective effect, at variance from wt Mag 2, suggesting that for Mag H2 the monolayer curvature could prevail over the peptide-membrane electrostatic interaction. We then mechanically characterized GUVs by measuring the effect of Mag H2 on the bending constant of lipid bilayers by flickering spectroscopy and, by using micropipette aspiration technique, we followed the steps leading to vesicle permeabilization. We found that Mag H2, notwithstanding its enhanced hydrophobicity, has a pore formation mechanism compatible with the toroidal pore model similar to that of wt Mag 2.

2019 - Role of Lipid Composition, Physicochemical Interactions, and Membrane Mechanics in the Molecular Actions of Microbial Cyclic Lipopeptides [Articolo su rivista]
Balleza, D.; Alessandrini, A.; Beltran Garcia, M. J.
abstract

Several experimental and theoretical studies have extensively investigated the effects of a large diversity of antimicrobial peptides (AMPs) on model lipid bilayers and living cells. Many of these peptides disturb cells by forming pores in the plasma membrane that eventually lead to the cell death. The complexity of these peptide–lipid interactions is mainly related to electrostatic, hydrophobic and topological issues of these counterparts. Diverse studies have shed some light on how AMPs act on lipid bilayers composed by different phospholipids, and how mechanical properties of membranes could affect the antimicrobial effects of such compounds. On the other hand, cyclic lipopeptides (cLPs), an important class of microbial secondary metabolites, have received comparatively less attention. Due to their amphipathic structures, cLPs exhibit interesting biological activities including interactions with biofilms, anti-bacterial, anti-fungal, antiviral, and anti-tumoral properties, which deserve more investigation. Understanding how physicochemical properties of lipid bilayers contribute and determining the antagonistic activity of these secondary metabolites over a broad spectrum of microbial pathogens could establish a framework to design and select effective strategies of biological control. This implies unravelling—at the biophysical level—the complex interactions established between cLPs and lipid bilayers. This review presents, in a systematic manner, the diversity of lipidated antibiotics produced by different microorganisms, with a critical analysis of the perturbing actions that have been reported in the literature for this specific set of membrane-active lipopeptides during their interactions with model membranes and in vivo. With an overview on the mechanical properties of lipid bilayers that can be experimentally determined, we also discuss which parameters are relevant in the understanding of those perturbation effects. Finally, we expose in brief, how this knowledge can help to design novel strategies to use these biosurfactants in the agronomic and pharmaceutical industries.

2018 - Effects of the peptide Magainin H2 on Supported Lipid Bilayers studied by different biophysical techniques [Articolo su rivista]
Marín-Medina, Nathaly; Mescola, Andrea; Alessandrini, Andrea
abstract

Given the increasing trend in bacterial antibiotic resistance, research on antimicrobial peptides and their mechanisms of action has become of huge relevance in the last years. Several studies have investigated the effects of a large variety of antimicrobial peptides directly on bacteria or on model lipid bilayers. In the case of model lipid bilayers, different systems are typically exploited; however, different results could be obtained due to the specific properties of the used system. Supported Lipid Bilayers and Giant Unilamellar Vesicles are among the most popular model systems. Here we used Atomic Force Microscopy and fluorescence microscopy to study the interaction of the antimicrobial peptide Magainin H2, an analog of Magainin 2 with increased hydrophobicity, on Supported Lipid Bilayers. We found that, for this kind of model bilayer, due to its strong interaction with the support, the lateral expansion of the membrane induced by the interaction with the peptides is initially inhibited and subsequently proceeds creating new bilayer regions with many defects. This scenario gives rise in Supported Lipid Bilayers to effects like initial increase of lateral pressure, formation of lipid tubes to release this increase, or development of bilayer regions with lower lipid density. Our results highlight that care should be given to the selected model system when studying and comparing the interaction of peptides with other lipid bilayer model systems.

2018 - Mechanical phenotyping of K562 cells by the Micropipette Aspiration Technique allows identifying mechanical changes induced by drugs [Articolo su rivista]
Di Cerbo, Alessandro; Rubino, Valentina; Morelli, Francesca; Ruggiero, Giuseppina; Landi, Rosaria; Guidetti, Gianandrea; Canello, Sergio; Terrazzano, Giuseppe; Alessandrini, Andrea
abstract

Mechanical properties of living cells can be used as reliable markers of their state, such as the presence of a pathological state or their differentiation phase. The mechanical behavior of cells depends on the organization of their cytoskeletal network and the main contribution typically comes from the actomyosin contractile system, in both suspended and adherent cells. In the present study, we investigated the effect of a pharmaceutical formulation (OTC - Ossitetraciclina liquida 20%) used as antibiotic, on the mechanical properties of K562 cells by using the Micropipette Aspiration Technique (MAT). This formulation has been shown to increase in a time dependent way the inflammation and toxicity in terms of apoptosis in in vitro experiments on K562 and other types of cells. Here we show that by measuring the mechanical properties of cells exposed to OTC for different incubation times, it is possible to infer modifications induced by the formulation to the actomyosin contractile system. We emphasize that this system is involved in the first stages of the apoptotic process where an increase of the cortical tension leads to the formation of blebs. We discuss the possible relation between the observed mechanical behavior of cells aspirated inside a micropipette and apoptosis.

2017 - AFM investigation of mechanical properties of glioblastoma multiforme cells and their relation to motility [Abstract in Atti di Convegno]
Mescola, A; Alessandrini, Andrea; Corsi, Lorenzo
abstract

Glial tumors are clinically classified in 4 groups according to their malignancy level. Glial tumors belonging to the IV group are called Glioblastoma Multiforme (GBM) and they are among the most aggressive brain tumors. In the recent years the mechanical phenotype of cells has been recognized as a valuable marker of their malignancy level [1-3]. Here we studied by AFM the mechanical behavior of U87mg cells when exposed to a drug which interferes with their cytoskeleton affecting also their migration ability. We found that U87mg cells exposed to the tested drug presented a decreased migration potential which is correlated with an increased stiffness of the cells and with a loss of polarity. By exploiting AFM Dynamic Mechanical Analysis we also characterized the behavior of the cells for different probing frequencies. By exploiting immunofluorescence microscopy we also investigated the effect of the tested drug on the reorganization of the cell cytoskeleton finding a strong increase of the presence of stress fibers.

2017 - Comment on "temperature fluctuations and the thermodynamic determination of the cooperativity length in glass forming liquids" (J. Chem. Phys. 146, 104501 (2017)) [Articolo su rivista]
Alessandrini, Andrea; Pieruccini, Marco; Tombari, Elpidio
abstract

No Abstract: comment

2017 - In vitro method for producing a flap of genetically modified cells on fibrin substrate [Brevetto]
Pellegrini, G.; Alessandrini, A.; De Luca, M.
abstract

2017 - Oxytetracycline induces DNA damage and epigenetic changes: A possible risk for human and animal health? [Articolo su rivista]
Gallo, Adriana; Landi, Rosaria; Rubino, Valentina; Di Cerbo, Alessandro; Giovazzino, Angela; Palatucci, Anna Teresa; Centenaro, Sara; Guidetti, Gianandrea; Canello, Sergio; Cortese, Laura; Ruggiero, Giuseppina; Alessandrini, Andrea; Terrazzano, Giuseppe
abstract

Background. Oxytetracycline (OTC), which is largely employed in zootechnical and veterinary practices to ensure wellness of farmed animals, is partially absorbed within the gastrointestinal tract depositing in several tissues. Therefore, the potential OTC toxicity is relevant when considering the putative risk derived by the entry and accumulation of such drug in human and pet food chain supply. Despite scientific literature highlights several OTC-dependent toxic effects on human and animal health, the molecular mechanisms of such toxicity are still poorly understood. Methods. Here, we evaluated DNA damages and epigenetic alterations by quantitative reverse transcription polymerase chain reaction, quantitative polymerase chain reac- tion, chromatin immuno-precipitation and Western blot analysis. Results. We observed that human peripheral blood mononuclear cells (PBMCs) expressedDNAdamage features (activation ofATMand p53, phosphorylation ofH2AX and modifications of histone H3 methylation of lysine K4 in the chromatin) after the in vitro exposure to OTC. These changes are linked to a robust inflammatory response indicated by an increased expression of Interferon (IFN)- and type 1 superoxide dismutase (SOD1). Discussion. Our data reveal an unexpected biological in vitro activity of OTC able to modify DNA and chromatin in cultured human PBMC. In this regard, OTC presence in foods of animal origin could represent a potential risk for both the human and animal health.

2016 - Direct electrical control of IgG conformation and functional activity at surfaces [Articolo su rivista]
Ghisellini, Paola; Caiazzo, Marialuisa; Alessandrini, Andrea; Eggenhöffner, Roberto; Vassalli, Massimo; Facci, Paolo
abstract

We have devised a supramolecular edifice involving His-tagged protein A and antibodies to yield surface immobilized, uniformly oriented, IgG-type, antibody layers with Fab fragments exposed off an electrode surface. We demonstrate here that we can affect the conformation of IgGs, likely pushing/pulling electrostatically Fab fragments towards/from the electrode surface. A potential difference between electrode and solution acts on IgGs' charged aminoacids modulating the accessibility of the specific recognition regions of Fab fragments by antigens in solution. Consequently, antibody-antigen affinity is affected by the sign of the applied potential: a positive potential enables an effective capture of antigens; a negative one pulls the fragments towards the electrode, where steric hindrance caused by neighboring molecules largely hampers the capture of antigens. Different experimental techniques (electrochemical quartz crystal microbalance, electrochemical impedance spectroscopy, fluorescence confocal microscopy and electrochemical atomic force spectroscopy) were used to evaluate binding kinetics, surface coverage, effect of the applied electric field on IgGs, and role of charged residues on the phenomenon described. These findings expand the concept of electrical control of biological reactions and can be used to gate electrically specific recognition reactions with impact in biosensors, bioactuators, smart biodevices, nanomedicine, and fundamental studies related to chemical reaction kinetics.

2016 - EC-STM/STS of Redox Metalloproteins and Co-factors [Capitolo/Saggio]
Alessandrini, Andrea; Facci, Paolo
abstract

This chapter is intended to review some of the main experimental and theoretical results in the field of single molecule characterization of the electron transport through redox adsorbates. Both redox metalloproteins and co-factors have been object of intense investigation by EC-STM/STS techniques. Particularly, the paradigmatic case of the redox metalloprotein azurin and of derivatized quinone molecules adsorbed on atomically flat gold provide a valuable insight into the potential of the technique and into the attainable level of understanding of the electron transfer phenomenon. The results are discussed critically in view of their impact on the field of Molecular Electronics.

2016 - Electron transfer in nanobiodevices [Articolo su rivista]
Alessandrini, Andrea; Facci, Paolo
abstract

The present tutorial is aimed at introducing the reader to the main aspects of electron transfer in nanobiodevices. Nanobiodevices are faced both from scientific and technological viewpoints and their particular implementation as electron transfer devices provides the opportunity of presenting fundamentals of electron transfer theory. Examples of implementations of stand alone devices, along with those involving reconfigurable set-ups based on an electrochemical scanning tunneling microscope, enable introducing heterogeneous electron transfer and electron transport theories in electrochemical environment. Specific cases of nanobiodevices involving redox metalloproteins are reported and experimental results are interpreted and discussed in view of the most recent theoretical advancements, in order to provide the reader with a comprehensive view of the results and promises in this exciting branch of nanotechnology.

2016 - Metalloprotein electronics [Capitolo/Saggio]
Alessandrini, A.; Facci, P.
abstract

2015 - Effect of neurosteroids on a model lipid bilayer including cholesterol: An Atomic Force Microscopy study [Articolo su rivista]
Sacchi, Mattia; Puja, Giulia; Vena, Giulia; Alessandrini, Andrea; Facci, Paolo; Balleza, Daniel
abstract

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happenswhen the cholesterol concentration is decreased. Interestingly, an isoform of Allo, Isoallopregnanolone (3β,5α -tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.

2015 - Effects of neurosteroids on a model membrane including cholesterol: A micropipette aspiration study [Articolo su rivista]
Balleza, Daniel; Sacchi, Mattia; Vena, Giulia; Galloni, Debora; Puja, Giulia; Facci, Paolo; Alessandrini, Andrea
abstract

Amphiphilic molecules supposed to affect membrane protein activity could strongly interact also with the lipid component of the membrane itself. Neurosteroids are amphiphilic molecules that bind to plasma membrane receptors of cells in the central nervous system but their effect on membrane is still under debate. For this reason it is interesting to investigate their effects on pure lipid bilayers as model systems. Using the micropipette aspiration technique (MAT), here we studied the effects of a neurosteroid, allopregnanolone (3α,5α-tetrahydroprogesterone or Allo) and of one of its isoforms, isoallopregnanolone (3β,5α-tetrahydroprogesterone or isoAllo), on the physical properties of pure lipid bilayers composed by DOPC/bSM/chol. Allo is a well-known positive allosteric modulator of GABAA receptor activity while isoAllo acts as a non-competitive functional antagonist of Allo modulation. We found that Allo, when applied at nanomolar concentrations (50-200 nM) to a lipid bilayer model system including cholesterol, induces an increase of the lipid bilayer area and a decrease of the mechanical parameters. Conversely, isoAllo, decreases the lipid bilayer area and, when applied, at the same nanomolar concentrations, it does not affect significantly its mechanical parameters. We characterized the kinetics of Allo uptake by the lipid bilayer and we also discussed its aspects in relation to the slow kinetics of Allo gating effects on GABAA receptors. The overall results presented here show that a correlation exists between the modulation of Allo and isoAllo of GABAA receptor activity and their effects on a lipid bilayer model system containing cholesterol.

2015 - Method for estimating the cooperativity length in polymers [Articolo su rivista]
Pieruccini, Marco; Alessandrini, Andrea
abstract

The problem of estimating the size of the cooperatively rearranging regions (CRRs) in supercooled polymeric melts from an analysis of the α-process in ordinary relaxation experiments is addressed. The mechanism whereby a CRR changes its configuration is viewed as consisting of two distinct steps: a reduced number of monomers reaches initially an activated state, allowing for some local rearrangement; then, the subsequent regression of the energy fluctuation may take place through the configurational degrees of freedom, thus allowing for further rearrangements on larger length scales. The latter are indeed those to which the well-known Donth's scheme refers. Local readjustments are described in the framework of a canonical formalism on a stationary ensemble of small-scale regions, distributed over all possible energy thresholds for rearrangement. Large-scale configurational changes, instead, are described as spontaneous processes. Two main regimes are envisaged, depending on whether the role played by the configurational degrees of freedom in the regression of the energy fluctuation is significant or not. It is argued that the latter case is related to the occurrence of an Arrhenian dependence of the central relaxation rate. Consistency with Donth's scheme is demonstrated, and data from the literature confirm the agreement of the two methods of analysis when configurational degrees of freedom are relevant for the fluctuation regression. Poly(n-butyl methacrylate) is chosen in order to show how CRR size and temperature fluctuations at rearrangement can be estimated from stress relaxation experiments carried out by means of an atomic force microscopy setup. Cases in which the configurational pathway for regression is significantly hindered are considered. Relaxation in poly(dimethyl siloxane) confined in nanopores is taken as an example to suggest how a more complete view of the effects of configurational constraints would be possible if direct measurements of temperature fluctuations were combined with the proposed analysis.

2015 - Protein-based transistors [Capitolo/Saggio]
Alessandrini, A.; Facci, P.
abstract

Introduction: A survey of proteins in nanobioelectronics The idea of using proteins to assemble hybrid electronic devices stems from molecular electronics [1] and, as such, it is intimately connected with the advent of nanosciences and nanotechnologies, dating back to the early 1990s. Since then, much technological effort but less scientific effort has been deployed to try to implement devices that take advantage of the peculiar features of proteins. A technologist’s standpoint is that of regarding proteins as self-contained, nanometer-sized functional units, highly specialized and efficient in performing a certain functional task. Their efficiency is traced back to the fact that, being active parts of living beings, proteins are taking advantage of billions of years of natural evolution in specializing towards a given activity. This way of thinking, which one can often encounter in ritten or spoken accounts, appears to be questionable in light of a rather less naïve understanding of the theory of evolution, but is perhaps a good enough starting point to understand the historical motivations which led to the remarkable interest of a interdisciplinary part of the scientific community in the use of proteins for assembling electronic devices. The other aspect motivating the interest in proteins as elements in electronic circuits is their size.

2015 - Small and large scale segmental motion in polymers: Estimating cooperativity length by ordinary relaxation experiments [Articolo su rivista]
Pieruccini, Marco; Alessandrini, Andrea; Sturniolo, Simone; Corti, Maurizio; Rigamonti, Attilio
abstract

We derive a suitable expression for estimating the size of the cooperatively rearranging regions (CRRs) in supercooled polymer melts by fitting data worked out by ordinary relaxation experiments carried out in isothermal conditions. As an example, the average CRR size in poly(n-butyl methacrylate) in proximity to the glass transition temperature is derived from a stress relaxation experiment performed by means of an atomic force microscopy setup. Good agreement is found with results in the literature derived from measurements of temperature fluctuations (the so-called Donth method). The temperature dependence of the CRR size is explored for poly(butadiene); in this case the segmental relaxation function is derived through a novel method for the analysis of the efficiency with which free induction decay echoes are refocused in 1H NMR experiments. It is found that the CRR size increases upon cooling. The results derived from the analysis of the NMR data are found to be in satisfactory agreement with those worked out from broadband dielectric spectroscopy data in the literature

2014 - Model Bio‐Membranes Investigated by AFM and AFS: A Suitable Tool to Unravel Lipid Organization and their Interaction with Proteins [Capitolo/Saggio]
Alessandrini, Andrea; Facci, Paolo
abstract

The study of the biological membrane has largely benefitted from the exploitation of model bilayer systems. These simplified models of the complex biological membrane composed of thousands of different types of molecules allow both to understand basic physical principles underlying the membrane functioning and to test new techniques that will be subsequently applied to biological membranes. Here we concentrate on one of the most used model systems for this kind of investigations: the Supported Lipid Bilayer (SLB). In particular, we analyze the possibilities of investigation offered by Atomic Force Microscopy and Spectroscopy (AFM/AFS) on this model system. We discuss the information that this techniques is able to provide on the phase behavior of the lipid bilayers and on the partitioning of membrane proteins relative to the bilayer lateral heterogeneity. We discuss also the possibility to characterize the mechanical properties of lipid bilayers on the nanometer scale lateral resolution.

2014 - Phase transitions in supported lipid bilayers studied by AFM [Articolo su rivista]
Alessandrini, Andrea
abstract

We review the capabilities of Atomic Force Microscopy (AFM) in the study of phase transitions in Supported Lipid Bilayers (SLBs). AFM represents a powerful technique to cover the resolution range not available to fluorescence imaging techniques and where spectroscopic data suggest what the relevant lateral scale for domain formation might be. Phase transitions of lipid bilayers involve the formation of domains characterized by different heights with respect to the surrounding phase and are therefore easily identified by AFM in liquid solution once the bilayer is confined to a flat surface. Even if not endowed with high time resolution, AFM allows light to be shed on some aspects related to lipid phase transitions in the case of both a single lipid component and lipid mixtures containing sterols also. We discuss here the obtained results in light of the peculiarities of supported lipid bilayer model systems.

2013 - Electrochemical scanning tunneling microscopy and spectroscopy for single-molecule investigation [Capitolo/Saggio]
Alessandrini, Andrea; Facci, Paolo
abstract

The technique of electrochemical scanning tunneling microscopy (ECSTM) and spectroscopy (ECSTS) for studying electron transport through single redox molecules is here described. Redox molecules of both biological and organic nature have been studied by this technique with the aim of understanding the transport mechanisms ruling the flow of electrons via a single molecule placed in a nanometer-sized gap between two electrodes while elucidating the role of the redox density of states brought about by the molecule. The obtained results provide unique clues to single-molecule transport behavior and support the concept of single-molecule electrochemical gating.

2013 - Hydroquinone-Benzoquinone Redox Couple as a Versatile Element for Molecular Electronics [Articolo su rivista]
Alessandrini, Andrea; Petrangolini, Paolo
abstract

The possibility of controlling electron transport in a single molecule bridged between two metal electrodes represents the ultimate goal of molecular electronics. Molecular electronics aims also at introducing specific properties for the electron transport features both by controlling the structural details of the junction and by exploiting new chemical functionalities. Here we show that, in a molecular junction, where electrodes are represented by a gold substrate and the tip of a scanning tunneling microscope in electrochemical environment, the use of a single molecular species makes it possible to obtain different features for the tunneling current according to the structural details of the junction. In particular, molecules endowed with redox properties brought about by a hydroquinone/benzoquinone redox couple can show both transistor-like and negative differential resistance (NDR) effects. We discuss the mechanistic processes that might describe the different behavior in light of theories of electron transfer between metal electrodes and redox molecules. The results show, on the one hand, the great potential and flexibility that molecular electronics offer and, on the other hand, the need of controlling as much as possible the details of the tunneling junction in order to obtain reproducible results.

2012 - Adhesion mechanisms of the contact interface of TiO2 nanoparticles in films and aggregates [Articolo su rivista]
S., Salameh; J., Schneider; J., Laube; Alessandrini, Andrea; P., Facci; J. W., Seo; L. C., Ciacchi; L., Mädler
abstract

Fundamental knowledge about the mechanisms of adhesion between oxide particles with diameters of few nanometers is impeded by the difficulties associated with direct measurements of contact forces at such a small size scale. Here we develop a strategy based on AFM force spectroscopy combined with all-atom molecular dynamics simulations to quantify and explain the nature of the contact forces between 10 nm small TiO2 nanoparticles. The method is based on the statistical analysis of the force peaks measured in repeated approaching/retracting loops of an AFM cantilever into a film of nanoparticle agglomerates and relies on the in-situ imaging of the film stretching behavior in an AFM/TEM setup. Sliding and rolling events first lead to local rearrangements in the film structure when subjected to tensile load, prior to its final rupture caused by the reversible detaching of individual nanoparticles. The associated contact force of about 2.5 nN is in quantitative agreement with the results of molecular dynamics simulations of the particle–particle detachment. We reveal that the contact forces are dominated by the structure of water layers adsorbed on the particles’ surfaces at ambient conditions. This leads to nonmonotonous force–displacement curves that can be explained only in part by classical capillary effects and highlights the importance of considering explicitly the molecular nature of the adsorbates.

2012 - Dynamic Force Spectroscopy on Supported Lipid Bilayers: Effect ofTemperature and Sample Preparation [Articolo su rivista]
Alessandrini, Andrea; H. M., Seeger; T., Caramaschi; P., Facci
abstract

Biological membranes are constantly exposed to forces. The stress-strain relation in membranes determines thebehavior of many integral membrane proteins or other membrane related-proteins that show a mechanosensitive behavior. Here, we studied by force spectroscopy the behavior of supported lipid bilayers (SLBs) subjected to forces perpendicular to their plane. We measured the lipid bilayer mechanical properties and the force required for the punch-through event characteristic of atomic force spectroscopy on SLBs as a function of the interleaflet coupling. We found that for an uncoupled bilayer, the overall tip penetration occurs sequentially through the two leaflets, giving rise to two penetration events. In the case of a bilayer with coupled leaflets, penetration of the atomic force microscope tip always occurred in a single step. Considering the dependence of the jump-through force value on the tip speed, we also studied the process in the context of dynamic force spectroscopy (DFS). We performed DFS experiments by changing the temperature and cantilever spring constant, and analyzed the resultsin the context of the developed theories for DFS. We found that experiments performed at different temperatures and withdifferent cantilever spring constants enabled a more effective comparison of experimental data with theory in comparisonwith previously published data.

2012 - Nanoscale mechanical properties of lipid bilayers and their relevance in biomembrane organization and function [Articolo su rivista]
Alessandrini, Andrea; P., Facci
abstract

The mechanical properties of biological systems are emerging as fundamental in determining their functional activity. For example, cells continuously probe their environment by applying forces and, at the same time, are exposed to forces produced by the same environment. Also in biological membranes, the activity of membrane related proteins are affected by the overall mechanical properties of the hosting environment. Traditionally, the mesoscopic mechanical properties of lipid bilayers have been studied by micropipette aspiration techniques. In recent years, the possibility of probing mechanical properties of lipid bilayers at the nanoscale has been promoted by the force spectroscopy potentiality of Atomic Force Microscopes (AFM). By acquiring force-curves on supported lipid bilayers (SLBs) it is possible to probe the mechanical properties on a scale relevant to the interaction between membrane proteins and lipid bilayers and to monitor changes of these properties as a result of a changing environment. Here, we review a series of force spectroscopy experiments performed on SLBs with an emphasis on the functional consequences the measured mechanical properties can have on membrane proteins. We also discuss the force spectroscopy experiments on SLBs in the context of theories developed for dynamic force spectroscopy experiments with the aim to extract the kinetic and energetic description of the process of membrane rupture.

2012 - γ-Hemolysin oligomeric structure and effect of its formation on supported lipid bilayers: An AFM Investigation. [Articolo su rivista]
Alessandrini, Andrea; G., Viero; M., Dalla Serra; G., Prévost; P., Facci
abstract

γ-Hemolysins are bicomponent β-barrel pore forming toxins produced by Staphylococcus aureus as water-soluble monomers, which assemble into oligomeric pores on the surface of lipid bilayers. Here, after investigating the oligomeric structure of γ-hemolysins on supported lipid bilayers (SLBs) by atomic force microscopy (AFM), we studied the effect produced by this toxin on the structure of SLBs. We found that oligomeric structures with different number of monomers can assemble on the lipid bilayer being the octameric form the most stable one. Moreover, in this membrane model we found that γ-hemolysins can form clusters of oligomers inducing a curvature in the lipid bilayer, which could probably enhance the aggressiveness of these toxins at high concentrations.

2011 - Changes in single K+ channel behavior induced by a lipid phase transition [Articolo su rivista]
Alessandrini, Andrea; Facci, Paolo
abstract

Membrane protein activity is affected by the properties of the lipid bilayer hosting them. These properties are established by both the lipid composition and the thermodynamic state of the bilayer. In the latter case, any parameter that can alter the state of the bilayer is indirectly able to affect the activity of membrane proteins. In a recent study, we have demonstrated that the activity of the KcsA ion channel is strongly related to the thermodynamic state of the lipid bilayer. In particular, when the lipid bilayer is in its main phase transition region, the conductivity of KcsA is increased and all its characteristic times change according to the characteristic times of the lipid bilayer. We propose here that the lipid bilayer can affect the distribution among many conformational substates of the open channel, affecting the corresponding channel conductivity.

2011 - Electron Transport Properties of Single-Molecule-Bearing Multiple Redox Levels Studied by EC-STM/STS [Articolo su rivista]
Petrangolini, Paolo; Alessandrini, Andrea; M. L., Navacchia; M. L., Capobianco; P., Facci
abstract

Multielectron systems as possible components of molecular electronics devices are attracting compelling experimental and theoretical interest. Here we studied by electrochemical scanning tunneling techniques (EC-STMicroscopy and EC-STSpectroscopy) the electron transport properties of a redox molecule endowed with two redox levels, namely, the hydroquinone/quinone (H2Q/Q) couple. By forming self-assembled monolayers on Au(111) of oligo-phenylene-vinylene (OPV) derivatized H2Q/Q moieties, we were able to explore the features of the tunneling current/overpotential relation in the EC-STS setup. The behavior of the tunneling current sheds light onto the mechanism of electron transport involving the redox levels of the H2Q/Q redox pair coupled to tip and substrate electrodes.

2011 - Palytoxin induces cell lysis by priming a two-step process in MCF-7 cells [Articolo su rivista]
Prandi, Simone; Sala, GIAN LUCA; Bellocci, Mirella; Alessandrini, Andrea; P., Facci; Bigiani, Albertino; Rossini, Gian Paolo
abstract

The cytolytic action of palytoxin (PlTX) was recognized long ago, but its features have remained largely undetermined. We used biochemical, morphological, physiological and physical tools, to study the cytolytic response in MCF-7 cells, as our model system. Cytolysis represented a stereotyped response induced by addition of isotonic phosphate buffer (PBS) to cells that had been exposed to PlTX, after toxin removal and under optimal and sub-optimal experimental conditions. Cytolysis was sensitive to osmolytes present during cell exposure to PlTX but not in the course of the lytic phase. Fluorescence microscopy showed that PlTX caused cell rounding and rearrangement of the actin cytoskeleton. Atomic force microscopy (AFM) was used to monitor PlTX effects in real time, and we found that morphological and mechanical properties of MCF-7 cells did not change during toxin exposure, but increased cell height and decreased stiffness at its surface were observed when PBS was added to PlTX-treated cells. The presence of an osmolyte during PlTX treatment prevented the detection of changes in morphological and mechanical properties caused by PBS addition to toxin-treated cells, as detected by AFM. By patch-clamp technique, we confirmed that PlTX action involved the transformation of the Na+,K+-ATPase into a channel, and found that cell membrane capacitance was not changed by PlTX, indicating that the membrane surface area was not greatly affected in our model system. Overall, our findings show that the cytolytic response triggered by PlTX in MCF-7 cells includes a first phase, that is toxin-dependent and osmolyte-sensitive, priming cells to lytic events taking place in a separate phase, that does not require the presence of the toxin and is osmolyte-insensitive, but is accompanied by marked reorganization of actin-based cytoskeleton and altered mechanical properties at the cell’s surface. A model of the two step process of PlTX-induced cytolysis is presented.

2011 - Unraveling lipid/protein interaction in model lipid bilayers by Atomic Force Microscopy [Articolo su rivista]
Alessandrini, Andrea
abstract

The current view of the biological membrane is that in which lipids and proteins mutually interact to accomplish membrane functions. The lateral heterogeneity of the lipid bilayer can induce partitioning of membrane-associated proteins, favoring protein-protein interaction and influence signaling and trafficking. The Atomic Force Microscope allows to study the localization of membrane-associated proteins with respect to the lipid organization at the single molecule level and without the need for fluorescence staining. These features make AFM a technique of choice to study lipid/protein interactions in model systems or native membranes. Here we will review the technical aspects inherent to and the main results obtained by AFM in the study of protein partitioning in lipid domains concentrating in particular on GPI-anchored proteins, lipidated proteins, and transmembrane proteins. Whenever possible, we will also discuss the functional consequences of what has been imaged by Atomic Force Microscopy.

2011 - What do we really measure in AFM punch-through experiments on supported lipid bilayers? [Articolo su rivista]
Alessandrini, Andrea; H. M., Seeger; A., Di Cerbo; T., Caramaschi; P., Facci
abstract

Nowadays, there is much experimental evidence that the mechanical properties of biological membranes affect membrane protein functions. A very convenient technique to study these properties on a spatial scale relevant to that of single proteins is represented by Atomic Force Spectroscopy (AFS). In this study we measured the force the AFM tip has to apply on a supported lipid bilayer to punch-through it as a function of different environmental parameters. We observed that this force is reduced when the lipid bilayer is in its phase transition region. We interpreted our results on the basis of thermodynamical considerations and we stressed their biological relevance. In particular, the reduced punch-through force in the phase transition region could be relevant for the function of membrane proteins which operates by conformational changes at the protein/lipid interface. We also suggest that the presence of a transmembrane voltage drop can affect the measured punch-through force.

2010 - An Electrochemical Scanning Tunneling Microscopy Study of 2-(6-Mercaptoalkyl)hydroquinone Molecules on Au(111) [Articolo su rivista]
PETRANGOLINI, PAOLO; ALESSANDRINI, Andrea; L., Berti; P., Facci
abstract

The hydroquinone/benzoquinone redox couple involves the exchange of two electrons and two protons in its oxidation/reduction reaction in aqueous buffered solutions. In this work, we employed Electrochemical Scanning Tunneling Microscopy and Spectroscopy (EC-STM, EC-STS) to study the interfacial electron transfer properties of hydroquinone incorporated in a Self Assembled Monolayer on a Au(111) substrate. The exchange of electrons between the STM tip and the substrate is regulated by the redox levels of the sandwiched molecule and showed the presence of two regions of tunneling enhancement in the tunneling current/overvoltage relationship. The two regions can be attributed to the presence of two one-electron transfer processes whose equilibrium positions shift upon pH variations. This is the first time a redox molecule involving the exchange of both electrons and protons is studied by EC-STM and EC-STS. The hydroquinone/benzoquinone redox couple can be exploited to obtain an electrochemically or a pH gated transistor.

2010 - Changes in Single K(+) Channel Behavior Induced by a Lipid Phase Transition [Articolo su rivista]
Hm, Segger; L., Aldrovandi; Alessandrini, Andrea; P., Facci
abstract

We show that the activity of an ion channel is correlated with the phase state of the lipid bilayer hosting the channel By measuring unitary conductance dwell times and open probability of the K(+) channel KcsA as a function of temperature in lipid bilayers composed of POPE and POPG in different relative proportions we obtain that all those properties show a trend inversion when the bilayer is in the transition region between the liquid disordered and the solid ordered phase These data suggest that the physical properties of the lipid Mayer influence ion channel activity likely via a fine tuning of its conformations In a more general interpretative framework we suggest that other parameters such as pH ionic strength, and the action of amphiphilic drugs can affect the physical behavior of the lipid bilayer in a fashion similar to temperature changes resulting in functional changes of transmembrane proteins

2010 - Supported Lipid Bilayers on Mica and Silicon Oxide: Comparison of the Main Phase Transition Behavior [Articolo su rivista]
H. M., Seeger; A., Di Cerbo; Alessandrini, Andrea; P., Facci
abstract

The usual biophysical approach to the study of biological membranes is that of turtling to model systems. From these models, general physical principles ruling the lateral membrane structure can be obtained. A promising model system is the supported lipid bilayer (SLB) which could foresee the simultaneous investigation of the structure and physical properties of lipid bilayers reconstituted with membrane proteins. A complete exploitation of the model system to retrieve biologically relevant information requires an in-depth knowledge of the possible effect that experimental parameters could have on the behavior of the SLB. Here we used atomic force microscopy (AFM) to study the effect of different types of substrates on the behavior of SLBs as far as their main phase transition is concerned. We found that different substrates (mica and silicon oxide) can affect in dissimilar ways the interleaflet coupling of the bilayer, which might represent a sort of lipid signaling allowing communication between receptors on the extracellular leaflet and cytoplasmic components. By decreasing the interaction between the SLB and the substrate the interleaflet coupling is preserved independently of the bilayer preparation strategy. Moreover, we investigated by time-lapse AFM an isothermal phase transition induced by a pH change on a SLB. We established that the presence of a pH gradient across the bilayer can weaken the strength of the interleaflet coupling which is present in symmetrical pH conditions.

2009 - A Highly Selective, Biofunctional Surface for Molecule/Cell Sorting [Articolo su rivista]
M., Caiazzo; Alessandrini, Andrea; P., Facci
abstract

We report in this paper an approach to the effective capture of IgM antibodies from antisera and solutions based on the formation of a carpet of molecules exposing thiols off a surface. Surfaces of different nature, such as OH-exposing (glass, SiO2, metal oxides, etc.) and noble metal ones (Au, Ag, etc.), have been first functionalized in the liquid phase by suitable chemistry [3-(mercaptopropyl)trimethoxysilane or 1,4-benzenedimethanethiol]. The resulting exposed SH moieties have been further used for binding anti-A, -B, and -D IgM molecules from goat sera via a thiol exchange reaction involving the J chain and other disulfide bonds present in the IgM molecular structure. Antibodies preserve their functional activity at the surface and appear to be able to bind specifically erythrocytes of the proper group in a fast and reliable way. These results can be generalized to the use of any kind of IgM antibody and can be valuable in surface biofunctionalization in the fields of biosensors and immunoassays.

2009 - A one-pot functionalization strategy for immobilizing proteins onto linear dsDNA scaffolds [Articolo su rivista]
L., Berti; I. L., Medintz; Alessandrini, Andrea; P. Facci, P.
abstract

Functional DNA scaffolds can be defined as DNA-based structures comprising chemical moieties facilitating and guiding the immobilization of additional nanocomponents. Due to the limited reactivity of DNA there is currently a need to develop rapid routes to expand its chemical repertoire and increase its versatility as a nanostructuring scaffold. We report a simple synthetic strategy for generating linear and stable double-stranded DNA scaffolds functionalized with multiple sites reactive towards free thiols, and the utility of this approach is demonstrated by immobilizing a model protein containing an accessible free thiol. This procedure is very versatile and could be easily expanded to other types of chemistries. This approach could also potentially be employed for the specific, oriented immobilization of various biomolecules and nanoparticles on predefined DNA architectures.

2009 - AFM and FTIR Spectroscopy Investigation of the Inverted Hexagonal Phase of Cardiolipin [Articolo su rivista]
ALESSANDRINI, Andrea; U., Muscatello
abstract

Atomic force microscopy (AFM) and FTIR spectroscopy techniques have been exploited to investigate the inverted hexagonal phase (HII) of cardiolipin obtained by dehydration of a phospholipid water dispersion on a solid support. The characteristic cylinders of the HII phase have been imaged by AFM and the effects of different preparation conditions (temperature and the presence of chemicals) on the structural parameters and on the presence of local nanoscale defects have been studied. It has been found that the measured repeat spacing of the HII cylinders decreases upon increase of temperature and addition of pentachlorophenol (PCP), a chemical which is known to affect the structure and function of lipid bilayers. It has been shown that AFM can help in revealing some features of the mechanism of the inverted hexagonal phase formation, corroborating the results of a recent molecular dynamics study on the HII phase formation from multilamellar phospholipid structures.

2009 - Effect of Physical Parameters on the Main Phase Transition of Supported Lipid Bilayers [Articolo su rivista]
H. M., Seeger; G., Marino; Alessandrini, Andrea; P., Facci
abstract

Supported lipid bilayers (SLB) composed of 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) and -phosphatidylglycerol (POPG) were assembled by the vesicle fusion technique on mica and studied by temperature-controlled AFM. The role of different physical parameters on the main phase transition was elucidated. Both mixed (POPE/POPG 3:1) and pure POPE bilayers were studied. By increasing the ionic strength of the solution and the incubation temperature, a shift from a decoupled phase transition of the two leaflets, to a coupled transition, with domains in register, was obtained. The observed behavior points to a modulation of the substrate/bilayer and interleaflet coupling induced by the environment and preparation conditions of supported lipid bilayers. The results are discussed in view of the role of different interactions in the system. The influence of the substrate on the lipid bilayers can help understanding the possible effect that submembrane elements like the cytoskeleton might have on the structure and dynamics of biomembranes also in terms of interleaflet coupling.

2009 - Phase-Transition-Induced protein redistribution in Lipid Bilayers [Articolo su rivista]
H. M., Seeger; Bortolotti, Carlo Augusto; Alessandrini, Andrea; P., Facci
abstract

We report an atomic force microscopy study on the lateral spatial redistribution of an integral membraneprotein reconstituted in supported lipid bilayers (SLBs) subjected to a thermally induced phase transition.KcsA proteins were reconstituted in proteoliposomes of POPE/POPG (3:1, mol/mol), and SLBs, includingthe proteins, were then obtained by the vesicle fusion technique on mica. By decreasing the temperature, thelipid bilayer passed from a liquid disordered (ld) phase in which the proteins are homogeneously distributedto a coexistence of solid ordered (so) and ld domains with the proteins preferentially distributed in the lddomains. The inhomogeneous distribution eventually led to protein clustering. The obtained results are discussedin light of the role that the lipid/protein interaction can have in determining the function of integral membraneproteins.

2008 - Electrochemically assisted scanning probe microscopy: A powerful tool in nano(bio)science [Capitolo/Saggio]
Alessandrini, A.; Facci, P.
abstract

Nanosciences, in general and nanobiophysics, in particular,have taken much advantage of the advent of scanning probemicroscopies. These instruments have enabled real space visualizationof atoms and molecules allowing the retrieval of unprecedentedlyaccurate information. Nevertheless, the most powerful implementationsof scanning probe microscopies should also enable a full controlof the phenomena taking place at solid–liquid interfaces (e.g., electrochemicalreactions). In this chapter, we will review the applicationsof scanning probe microscopies (STM and SFM) under electrochemicalcontrol. In particular, we will firstly present the fundamentals ofelectrochemically controlled scanning tunnelling microscopy, as faras basic concepts and possible set-ups are concerned, and proceedto the basic applications involving characterization of clean surfaces,study of underpotential deposition of metals, potential-induced phasetransitions in molecular layers. The further paragraphs will be devotedto reviewing biophysical applications of electrochemical scanning tunnellingmicroscopy (EC-STM) as far as investigation on redox metalloproteinsis concerned. A discussion on the state-of-the-art videorate EC-STM is provided. In the last sections, we will present the currentand future efforts aimed at further developing electrochemicallyassisted scanning probe microscopy towards the implementation of anelectrochemically controlled current sensing atomic force microscopewith the fundamental contribution of state-of-the-art nanotechnology.

2008 - Imparting chemical specificity to nanometer-spaced electrodes [Articolo su rivista]
ALESSANDRINI, Andrea; L., Berti; Gazzadi, G. C.; P., Facci
abstract

In this paper we are demonstrating an electrochemically driven self-assembling approach to achieve the space-resolved chemical functionalization of nanoelectrodes. After forming a self-assembled monolayer of electroactive quinones on a pair of nano-spaced (< 100 nm) electrodes, we enabled the binding of ssDNA exclusively on a single nanoelectrode by controlling the oxidation state at each modified electrode. This procedure attained the chemical differentiation of otherwise identical nanoelectrodes as the immobilized ssDNA retained its hybridization ability. Furthermore, we established that Kelvin probe force microscopy is a suitable space-resolved analytical technique for detecting this chemical functionalization at the nanoscale. The reported approach, enabling the space-selective patterning of (bio)molecules on nanoelectrode surfaces, can find application in complex nanosensor structure and molecular electronics implementations.

2008 - Potential-induced morphological modifications in Xenopus laevis oocyte membranes containing KAT1 hexogenous K+ channels studied by electrochemical scanning force microscopy [Articolo su rivista]
Alessandrini, Andrea; P., Gavazzo; C., Picco; P., Facci
abstract

We report on a novel use of electrochemical scanning force microscopy (SFM) for the investigation of morphological modifications occurring in plasma membranes containing voltage-gated ion channels, on membrane potential variation. Membrane patches of Xenopus laevis oocytes microinjected with exogenous KAT1 cRNA, deposited by a stripping method at the surfaceof a derivatized gold film in inside-out configuration, have been imaged by SFM in an electrochemical cell. A potentiostat was used to maintain a desired potential drop across the membrane. Performing imaging at potential values corresponding to open (2120 mV) and closed (120 mV) states for KAT1, morphological differences in localized sample zones were observed. Particularly, crossshaped features involving a significant membrane portion appear around putative channel locations. The reported approach constitutes the first demonstration of an SPM-based experimentaltechnique suitable to investigate the rearrangements occurring to the plasma membrane containing voltage-gated channels on transmembrane potential variation.

2008 - Ultraflat Nickel Substrates for Scanning Probe Microscopy of Polyhistidine-Tagged Proteins [Articolo su rivista]
Alessandrini, Andrea; Bortolotti, Carlo Augusto; G., Bertoni; A., Vezzoli; P., Facci
abstract

A novel approach to the preparation of ultra-flat Nickel substrates suitable to Scanning Probe Microscopy imaging of immobilized polyhistidine-tagged proteins has been devised. Exploiting freshly cleaved mica, Ni thermal evaporation followed by thermal annealing in vacuum, and the template stripping method, we have obtained Ni substrates with a rms roughness as low as 0.12 nm, which bind readily polyhistidine-tagged proteins, enabling molecular resolution imaging of isolated molecules as well as of molecular sub-monolayers. Protein sample exposure to imidazole causes removal of the adsorbates, confirming the involvement of the poly-histidine tail in protein surface immobilization.

2007 - A reactive peptidic linker for self-assembling hybrid quantum dot-DNA bioconjugates [Articolo su rivista]
I. L., Medintz; L., Berti; T., Pons; A. F., Grimes; D. S., English; Alessandrini, Andrea; P., Facci; H., Mattoussi
abstract

Self-assembly of proteins, peptides, DNA, and other biomolecules to semiconductor quantum dots (QD) is an attractive bioconjugation route that can circumvent many of the problems associated with covalent chemistry and subsequent purification. Polyhistidine sequences have been shown to facilitate self-assembly of proteins and peptides to ZnS-overcoated CdSe QDs via complexation to unoccupied coordination metal sites on the nanocrystal surface. We describe the synthesis and characterization of a thiol-reactive hexahistidine peptidic linker that can be chemically attached to thiolated-DNA oligomers and mediate their self-assembly to CdSe-ZnS core-shell QDs. The self-assembly of hexahistidine-appended DNA to QDs is probed with gel electrophoresis and fluorescence resonance energy transfer techniques, and the results confirm high-affinity conjugate formation with control over the average molar ratio of DNA assembled per QD. To demonstrate the potential of this reactive peptide linker strategy, a prototype QD-DNA-dye molecular beacon is self-assembled and tested against both specific and nonspecific target DNAs. This conjugation route is potentially versatile, as altering the reactivity of the peptide linker may allow targeting of different functional groups such as amines and facilitate self-assembly of other nanoparticle-biomolecule structures.

2007 - Defects in ordered aggregates of cardiolipin visualized by atomic force microscopy [Articolo su rivista]
Alessandrini, Andrea; Valdre, G; Valdre, U; Muscatello, Umberto
abstract

The formation and the nature of defects in ordered aggregates of cardiolipin (tetra acyl di phosphatidyl glycerol) supported on solid substrates have been investigated by atomic force microscopy (AFM). The experiments were performed on two model systems, i.e. three-dimensional liquid crystals dispersed in water and partially de-hydrated on a hydrophilic surface, and two-dimensional films of molecules self-assembled onto an isotropic hydrophobic surface. Defects were induced both by varying the preparation temperature and by treatment with specific chemicals known to modify the order parameters in natural and artificial membranes, specifically: 2,4-dinitro-phenol (DNP) and pentachloro-phenol (PCP). The effect of lipid oxidation on the nanocrystalline order was also investigated. The images obtained by AFM allow to characterize the type of defects and their local density at nanoscale level. They also provide additional information to differentiate the specific role of acyl chains and polar heads in the process of lipid self-organization. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

2007 - Fine-Tuning Nanoparticle Size by Oligo(guanine)n Templated Synthesis of CdS: an AFM study [Articolo su rivista]
L., Berti; Alessandrini, Andrea; M., Bellesia; P., Facci
abstract

We are presenting a method for modulating the size of US nanoparticles by templating their formation with oligo(guanine)(n) oligomers where n varied from 5 to 20. The variation in template length resulted in observable changes in the size distribution of the US nanoparticles. Statistical analysis of AFM images showed a general trend whereby the US average height decreased for longer oligoG(n) and increased for shorter oligoG(n). Concomitantly, shorter oligoG(n). yielded more dispersed populations, while longer oligoGn gave less dispersed populations. This synthetic methodology could be extended to the synthesis of other nanoparticles and even to mixed-metal nanoparticles resulting in a powerful method for fine-tuning size-dependent properties.

2006 - A CMOS, fully integrated sensor for electronic detection of DNA hybridization [Articolo su rivista]
M., Barbaro; A., Bonfiglio; L., Raffo; Alessandrini, Andrea; P., Facci; I., Barak
abstract

An integrated field-effect device for fully electronic deoxyribonucleic acid (DNA) detection was realized in a standard CMOS process. The device is composed of a floating-gate MOS transistor, a control-capacitor acting as integrated counterelectrode, and an exposed active area for DNA immobilization. The drain-current of the transistor is modulated by the electric charge carried by the DNA molecules. After DNA hybridization, this charge increases and a change in the output current is measured. Experimental results are provided. Full compatibility with a standard CMOS process-opens the way to the realization of low-cost large-scale integration of fast electronic DNA detectors.

2006 - Controlled DNA-templated metal deposition: towards ultrathin nanowires [Articolo su rivista]
L., Berti; Alessandrini, Andrea; C., Menozzi; P., Facci
abstract

In this paper, we report the metallization of a dsDNA template using a novel photography-derived two-step strategy in which dsDNA is first complexed with Ag(I) ions and then irradiated with UV light at 254 nm. The nucleobases act as light harvesters and sensitizers, triggering the photoreduction of the complexed silver ions. This process yields a silver nanoparticles blueprint along the DNA strand. The silver latent image is then developed by depositing metallic nickel through an electroless plating process. This photography-derived procedure generates very homogeneous and evenly distributed strings of silver-core/nickel-shell nanoparticles. Although still discontinuous, we believe that such chains can serve as the base for obtaining continuous metal nanowires. Furthermore, this process can most likely be extended to other plating metals, resulting in a broadly general procedure for metallizing DNA with a variety of different materials. Because of the intrinsic simplicity in using light as the key step, this methodology might be amenable to large-scale development, eventually leading to a very efficient molecular-photolithography process.

2006 - Demonstration of an electrostatic-shielded cantilever [Articolo su rivista]
P., Pingue; V., Piazza; P., Baschieri; C., Ascoli; C., Menozzi; Alessandrini, Andrea; P., Facci
abstract

The fabrication and performances of cantilevered probes with reduced parasitic capacitance starting from a commercial Si3N4 cantilever chip is presented. Nanomachining and metal deposition induced by focused ion beam techniques were employed in order to modify the original insulating pyramidal tip and insert a conducting metallic tip. Two parallel metallic electrodes deposited on the original cantilever arms are employed for tip biasing and as ground plane in order to minimize the electrostatic force due to the capacitive interaction between cantilever and sample surface. Excitation spectra and force-to-distance characterization are shown with different electrode configurations. Applications of this scheme in electrostatic force microscopy, Kelvin probe microscopy and local anodic oxidation is discussed.

2006 - Fully electronic DNA hybridization detection by a standard CMOS biochip [Articolo su rivista]
M., Barbaro; A., Bonfiglio; L., Raffo; Alessandrini, Andrea; P., Facci; I., Barak
abstract

A novel solid-state biosensor for label-free detection of DNA hybridization is presented. The new device is realized in a standard CMOS process, thus allowing the realization of low-cost, portable, fully integrated devices. The detection mechanism is based on the field-effect of the intrinsic negative electric charge of DNA molecules which modulates the threshold voltage of a floating-gate MOS transistor. A fluid cell was developed for delivering DNA samples on the active surface of the chip. The device has an integrated, individual counter-electrode, so dry measurements are possible increasing lifetime of the chip and speeding up the experiment. Successful measurements on a first prototype of the chip, hosting 16 sensors individually addressable, are provided as proof of concept.

2006 - Monitoring cell-cycle-related viscoelasticity by a quartz crystal microbalance [Articolo su rivista]
Alessandrini, Andrea; Croce, Maria Antonietta; Tiozzo, Roberta; Facci, P.
abstract

We have monitored viscoelasticity variation of a cell population during the cell cycle by a Quartz Crystal Microbalance (QCM). Balb 3T3 fibroblasts were synchronized in the G0/G1 phase and seeded in a QCM chamber placed in a cell incubator. After cell sedimentation, the frequency signal was characterized by an amplitude modulation attributed to the viscoelasticity variation of the cells proliferating in phase. A control experiment with nonsynchronized cells showed a similar signal trend, but without significant modulation. Interestingly, the system resulted also to perform as a device sensitive to the effect of drugs affecting the cell cycle, such as colchicine.

2006 - Unravelling single metalloprotein electron transfer by scanning probe techniques [Articolo su rivista]
Alessandrini, Andrea; S., Corni; P., Facci
abstract

This review is intended to account for the experimental and theoretical achievements obtained in a period of about 15 years on the investigation of the electron transport through single redoxmetalloproteins by scanning probe techniques. A highly focussed research effort has been deployed by the scientists active in this particular field towards measuring and interpreting electronic current signals flowing via blue copper, redox metalloproteins (e.g. azurin). The field has taken a remarkable advantage of the use of electrochemically assisted scanning tunnelling microscope (EC-STM) which has allowed to probe single molecule signals under full control of all the potential values involved in the experiments. This experimental activity has both triggeredmore comprehensive theoretical interpretations and has been, in its turn, stimulated by theoreticians to test always new predictions. The authors hope to have succeeded in providing the reader with a valuable appraisal of this fascinating field.

2005 - AFM: a versatile tool in biophysics [Articolo su rivista]
Alessandrini, Andrea; P., Facci
abstract

Here we review the applications of atomic force microscopy to the study of samples of biological origin. Emphasis is given to provide the reader with information on the broad range of different biophysical applications that, to date, such a technique can deal with. After recalling briefly the operating principles of an atomic force microscope, the broad field of bio-imaging applications is faced (DNA, DNA-protein interaction, proteins, lipid membranes, cells); thereafter, the use of the atomic force microscope to measure forces is introduced and force mapping on living cells is discussed. This section is followed by the description of the use of force curves in assessing single-molecule inter- and intramolecular interactions. A paragraph on the perspectives of the technique in biophysical applications concludes the paper. We hope that this review can help the reader in appreciating how atomic force microscopy contributes to the current explosive growth of nanobiosciences, where biology, chemistry and physics merge.

2005 - Chemically homogeneous, silylated surface for effective DNA binding and hybridization [Articolo su rivista]
Alessandrini, Andrea; DE RENZI, Valentina; L., Berti; I., Barak; P., Facci
abstract

We report on a method for covalent immobilization of 5 ´-thiol-modified single strand DNA probes, onto oxygen exposing surfaces by exploiting surface derivatization by 3-mercaptopropyltrimethoxysilane and subsequent intermolecular disulfide bond formation. The various steps in the formation of the molecular edifices have been characterized by X-ray photoelectron spectroscopy, quartz crystal microbalance and atomic force microscopy under liquid. Surface reaction kinetics of thiol-modified DNA probes with thiol-bearing silanes turned out to be a second-order one, possibly due to the presence of both free thiol and S-S dimers in solution. The ability of immobilized single strand DNA to bind the complementary strand has been tested and confirmed by quartz crystal microbalance measurements. The presented DNA immobilization method appears to be applicable to any surface bearing exposed hydroxyl moieties.

2005 - DNA-templated photoinduced silver deposition [Articolo su rivista]
L., Berti; Alessandrini, Andrea; P., Facci
abstract

We are presenting a photography-derived methodology to achieve the photoreduction of Ag+−DNA complexes. λ-Phage DNA was first loaded with silver ions, then irradiated with UV light at 254 nm. The DNA bases acted as light sensitizers, promoting the in situ reduction of Ag+ and the formation of metallic silver clusters. Three different approaches will illustrate this procedure, and silver nanoparticle chains will be grown along a DNA template in a rapid and specific way.

2005 - Focused ion beam-nanomachined probes for improved electric force microscopy [Articolo su rivista]
C., Menozzi; Gazzadi, G. C.; Alessandrini, Andrea; P., Facci
abstract

Nanomachining and beam-assisted Pt deposition by a focused ion beam (FIB) was used to modify AFM probes for improved electric force measurements.. Si3N4 cantilevers have been endowed with a nano-electrode at the tip apex to confine the electro-sensitive area at the very tip. This action results in both a marked decrease of the parasitic capacitive effect and in an improved electric force microscopy (EFM) contrast and resolution, with respect to usual, full metal-coated cantilevers. This fabrication approach is suited to the development of innovative electro-sensitive probes, useful in different scanning probe techniques.

2005 - Single-metalloprotein wet biotransistor [Articolo su rivista]
Alessandrini, Andrea; Salerno, M.; Frabboni, Stefano; Facci, P.
abstract

Metalloproteins are redox molecules naturally shuttling electrons with high efficiency between molecular partners. As such, they are candidates of choice for bioelectronics. In this work, we have used bacterial metalloprotein azurin, hosted in a nanometer gap between two electrically biased gold electrodes, to demonstrate an electrochemically gated single-molecule transistor operating in an aqueous environment. Gold-chemisorbed azurin shows peaks in tunneling current upon changing electrode potential and a related variation in tunneling barrier transparency which can be exploited to switch an electron current through it. These results suggest the wet approach to molecular electronics as a viable method for exploiting electron transfer of highly specialized biomolecules. ©2005 American Institute of Physics

2004 - Grabbing Yeast iso-1-cytochrome c by Cys102: an effective approach for the assembly of functionally active metalloprotein carpets [Articolo su rivista]
Gerunda, Mimmo; Bortolotti, Carlo Augusto; Alessandrini, Andrea; Sola, Marco; Battistuzzi, Gianantonio; P., Facci
abstract

We report an approach for immobilizing iso-l-cytochrome c from Saccharomyces cerevisiae on oxygen exposing surfaces derivatized with SH-terminated silanes. The SH moieties from silanes have been brought to react with the partially buried Cys102, forming an intermolecular disulfide bond which anchored covalently cytochrome c to the surface. The presence of a single cysteine residue on the protein surface imparted a well-defined orientation to the molecular edifice. Molecular constructs obtained with native cytochrome c and with a cysteine-depleted mutant (C102T) have been investigated by means of scanning force microscopy under liquid, which was performed to assay the quality of the molecular carpet, showing that the native protein formed a robust monolayer at the surface, whereas only a negligible amount of physisorbed molecules were detected in the case of a mutant. UV-vis absorption spectroscopy was performed to confirm that immobilization takes place via the Cys102 residue. Linear sweep voltammetric measurements showed retention of the redox activity of the covalently immobilized cytochrome c, confirming the viability of the proposed immobilization method for obtaining monolayers of redox active molecules.

2003 - Electron tunnelling through azurin is mediated by the active site Cu ion [Articolo su rivista]
Alessandrini, Andrea; M., Gerunda; Canters, G. W.; Verbeet, M. P.; P., Facci
abstract

Cu- and Zn-azurin chemisorbed on Au(111) have been comparatively investigated by electrochemical scanning tunnelling microscopy in buffer solution. Cu-azurin shows a marked tunnelling current resonance upon substrate potential at -0.21 V (vs SCE), whereas Zn counterparts do not. These data, discussed in terms of current theories on electron tunnelling through redox adsorbates, demonstrate the role of the electroactive metal ion present in the active site in assisting electron transfer via this metalloprotein,

2003 - Lack of molecular relationships between lipid peroxidation and mitochondrial DNA single strand breaks in isolated rat hepatocytes and mitochondria [Articolo su rivista]
Scotti, C; Iamele, L; Alessandrini, Andrea; Vannini, V; Cazzalini, O; Lazze, Mc; Melli, R; Savio, M; Pizzala, R; Stivala, La; Biglieri, S; Tomasi, Aldo; Bianchi, L.
abstract

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo. (C) 2003 Elsevier Science B.V. and Mitochondria Research Society. All rights reserved.

2003 - Tuning molecular orientation in protein films [Articolo su rivista]
Alessandrini, Andrea; M., Gerunda; P., Facci; B., Schnyder; R., Kotz
abstract

The rational design of azurin metalloprotein assemblies suitable for biomolecular electronics application has been achieved by exploiting different surface chemical approaches for growing active protein layers on both metal and insulating surfaces. The formed layers, which have been tested extensively by scanning force microscopy (SFM), spectroscopic ellipsometry, X-ray photoelectron spectroscopy (XPS), and electrochemical measurements (cyclic and linear voltammetry (CV and LV)), consist of redox active molecules endowed with tuneable orientation according to the particular functional group exploited for surface immobilization. The peculiar molecular arrangement has turned out to be responsible for different transport properties in solid state hybrid electronic planar devices.

2003 - Work function dependence on the thickness and substrate of carbon contamination layers by Kelvin probe force microscopy [Articolo su rivista]
Alessandrini, A.; Valdre, U.
abstract

The contact potential difference (CPD) between carbon contamination (CC) layers and the several substrates on which they were deposited has been measured as a function of the film thickness by means of Kelvin probe force microscopy (KPFM). The observed CPD trends may be divided into three categories: (i) an increase, or decrease, in CPD with thickness up to a saturation value with sign inversion with respect to the substrates (Al and Si); (ii) an oscillation with no sign inversion (substrates, gold and platinum); (iii) an oscillation through sign inversion (palladium substrate). Effects (ii) and (iii) seem to be typical of CC, since they have not been observed for other materials, including evaporated carbon. Several possible causes of the above two effects are examined, but a satisfactory interpretation has not been found yet. The sensitivity of KPFM is such that CC layers 10 nm thick are easily visible, whereas they are hardly detectable by topography.

2002 - Atomic Force Microscopy: a modern technique in Membrane Science [Capitolo/Saggio]
Muscatello, Umberto; Alessandrini, Andrea; Valdre', G.; Valdre', U.
abstract

Atomic force microscopy: a modern versatile technique in membrane science

2002 - Electric force microscopy investigation of the microstructure of thick film resistors [Articolo su rivista]
Alessandrini, Andrea; G., Valdre'; Morten, Bruno; Prudenziati, Maria
abstract

Maps of the electric field distribution on the surface of thick film resistors (TFRs) have been acquired by using electric force microscopy (EFM). TFRs based on various types of conducting phases (Bi-ruthenate, Pb-ruthenate, or RuO2) and with different volume fractions in the glassy matrix have been examined. Scanning electron microscopy, x-ray energy dispersive spectroscopy and x-ray diffraction have been used to correlate the EFM results to the morphological, microchemical, and structural characteristics of the samples. The evolution of the TFRs microstructure and the segregation characteristic with the firing conditions have been investigated. The results showed that the concentration of the electric field around the conductive grains is a general feature of all the films, independently of the resistor composition, and a meander-like path of charge carriers on a microscopic scale has been assessed. The observations also indicated that at high firing temperatures the segregated structure did not disappear, but on the contrary was enhanced. All the results are critically discussed in relation to the electrical and piezoresistive properties of the TFRs and suggestions for new models to correlate the microstructure and the electric properties are presented.

2001 - Early effects of AZT on mitochondrial functions in the absence of mitochondrial DNA depletion in rat myotubes [Articolo su rivista]
Cazzalini, O.; Lazze, M. C.; Iamele, L.; Stivala, L. A.; Bianchi, L.; Vaghi, P.; Cornaglia, A.; Calligaro, A.; Curti, D.; Alessandrini, A.; Prosperi, E.; Vannini, V.
abstract

Zidovudine (AZT) is a potent inhibitor of human immunodeficiency virus (HIV) replication. In humans, as well as in animal models, long-term treatment with AZT induces a severe myopathy characterised by structural and functional alterations of mitochondria associated with depletion of mitochondrial DNA (mtDNA). In the present work, we compared the effects induced by AZT on mitochondria upon short- or long-term treatments of cultured rat myotubes. Morphological alterations were investigated by electron microscopy, and mtDNA depletion and deletions were analysed by Southern blot. Mitochondrial membrane potential was determined after JC-1 staining by laser-scanning confocal microscopy in whole cells, and by flow cytometry in isolated muscle mitochondria. We found that the early effects of AZT on mitochondrial functions were a marked, yet reversible reduction in mitochondrial membrane potential, in the absence of any effect on mtDNA. The long-term treatment, in addition to mitochondrial membrane potential alterations, induced morphological changes in mitochondria, and a remarkable reduction in the amount of mtDNA, without any significant evidence of mtDNA deletions. In both treatments, a block of the spontaneous contraction of myotubes was observed. To study in more detail the early effects induced by AZT, the ability of the drug to interact with cardiolipin, an important component of internal mitochondrial membrane, was investigated by atomic force microscopy (AFM) in an artificial membrane model system. The results suggest that the primary effects of AZT may be related to a physical interference with the membrane structure leading to a consequent modification of its physical characteristics. © 2001 Elsevier Science Inc. All rights reserved.

2001 - Study of the contrast in electric force microscopy images of RuO2-based thick-film resistors [Articolo su rivista]
Alessandrini, A.; Valdre, G.
abstract

The contrast mechanism of electric force microscopy (EFM) operating in static and dynamic modes have been investigated and applied to the clarification of the electrical conduction properties of RuO2-based thick-film resistors. Both the magnetic and the electrical contributions to the overall EFM signal and the corresponding contrast have been analysed and compared by using different types of atomic force microscopy tip (with a magnetic coating and with a Pt/Ir coating). It has been found that the EFM contrast changes on inverting the voltage polarity of the samples. The regions surrounding the RuO2 grains present an EFM signal which is lower for a negative bias than for a positive bias at low values of the applied voltage; this signal difference tends to disappear on increasing the absolute bias value. This behaviour, typical of semiconductors, ascribes to the above regions semiconducting properties.

2000 - Different patterns of collagen-proteoglycan interaction: A scanning electron microscopy and atomic force microscopy study [Articolo su rivista]
Raspanti, M.; Congiu, T.; Alessandrini, A.; Gobbi, P.; Ruggeri, A.
abstract

The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.

2000 - Lipid oxidation deletes the nanodomain organization of artificial membranes [Articolo su rivista]
Muscatello, Umberto; A., Alessandrini; G., Valdre; V., Vannini; U., Valdre
abstract

Nanoscopic domains with different crystal structures have been induced in closed artificial membranes and have been directly imaged by atomic force microscopy at a spatial resolution better than 0.3 nm. These observations provide experimental evidence to the hydrophobic mismatching theory of lateral phase separation phenomena. Under oxidant conditions, the lipid-lipid assembly reorganises into a new steady-state structure with disappearance of specific nanodomains. This finding may contribute to understanding the mechanism of peroxidative damage to membrane properties. In fact, alterations of specific anodes of molecular conformation and packing may lead to perturbation of specific properties.

1999 - Correlation between electric force microscopy and scanning electron microscopy for the characterization of percolative conduction in electronic devices [Articolo su rivista]
Alessandrini, Andrea; G., Valdre'; Morten, Bruno; S., Piccinini; Prudenziati, Maria
abstract

The correlation betweem electric force microscopy (EFM) and scanning electron microscopy (SEM)techniques provide interpretation of the contrast features of electric force images and gave an insight into the two-dimensional (2D) electrical transport properties of RuO2-based thick film resisitors (TFRs). From the comparison between EFM. tapping mode-atomic force microscopy, SEM (secondary electrons, specimen current and X-ray energy-dispersive spectroscopy) of TFRs on the same specimen area, it turned out that the conduction mechanism was related to the grain size and to how RuO2 crystals were distributed in the glassy matrix. Dark area in EFM images corresponded to conductive-insulating interfaces. In SEM the same areas were the interfaces between the Ru=2 crystals and the insulating matrix. The 2D percolative path of the electrical current near the surface was observed in connection with the net of RuO2 grains not homogeneously dispersed in the insulating matrix.

1999 - Correlation between electric force spectroscopy and scanning electron microscopy for the characterization of percolative conduction in electronic devices [Articolo su rivista]
Alessandrini, Andrea; G., Valdre'; Morten, Bruno; S., Piccinini; Prudenziati, Maria
abstract

The correlation between electron force microscopy (EFM) and SEM techniques provided interpretation of the contrast features of electron force images and gave an insight into the two-dimensional (2D) electron transport properties of RuO2-based thick film resistors (TFRs). From the comparison between EFM, tapping-mode atomic force microscopy, SEM (secondary electrons, specimen current and x-ray energy-dispersive spectroscopy) of TFRs on the same specimen area, the conduction mechanism was related to the grain size and to how RuO2 crystals were distributed in the glassy matrix. Dark areas in EFM images corresponded to conductive-insulating interfaces. In SEM the same areas were the interfaces between the RuO2 crystals and the insulating matrix. The 2-dimensional percolative path of the electron current near the surface was observed in connection with the net of RuO2 grains not homogeneously dispersed in the insulating matrix.

1999 - Nitrogen sorption tests, SEM-windowless EDS and XRD analysis of mechanically alloyed nanocrystalline getter materials [Articolo su rivista]
Valdre, G.; Zacchini, D.; Berti, R.; Costa, A.; Alessandrini, A.; Zucchetti, P.; Valdre, U.
abstract

Gas absorbing materials (getters) find several applications in modern vacuum technology; in particular to maintain required vacuum levels in evacuated and sealed enclosures. The gas absorbing properties of these getters depend on the physico-chemical nature of their surfaces. The aim of this work is to study the absorption properties of commercial Zr-based alloys (non-evaporable getters) after mechanical alloying by means of a high vacuum planetary ball milling equipped with an in-situ compaction facility. The main aim was to refine the grain size and to develop particular defect structures to enhance the getter properties. The results have shown an improvement of the specific pumping speed of the ball milled commercial Zr-Zr(V,Fe)2 alloy with respect to the starting microcrystalline material. In particular, under our experimental conditions, the specific pumping speed vs absorbed gas curve presents a maximum after 2 hours of milling; prolonged milling reduced the pumping speed of the alloyed material. This behaviour is explained in terms of two opposing simultaneous chemical and structural effects.

1998 - Study of elastic fiber organization by scanning force microscopy [Articolo su rivista]
Ronchetti, Ivonne; Alessandrini, Andrea; Contri, Miranda; Fornieri, Claudio; Mori, Giuseppe; Quaglino, Daniela; U., Valdré
abstract

Elastic fibers of beef ligamentum nuchae were observed by atomic force microscopy and data compared with those obtained by conventional and freeze-fracture electron microscopy. Fresh isolated elastin fibers as well as thin sections of ligament fragments, which were fixed and embedded either in relaxed or in stretched, conditions, were analysed. The results confirm that, at least in beef ligamentum nuchae, elastic fibers consist of beaded filaments which can be oriented by stretching in the direction of the force applied. Moreover, atomic force microscopy revealed that these beaded filaments are laterally connected by periodical bridges which become more pronounced upon stretching. The data clearly show that elastin molecules are organized in a rather ordered array, at least at the super-molecular level, and a depiction of the elastin organization in beef ligament um nuchae is attempted.

1997 - Direct visualization of collagen-bound proteoglycans by tapping-mode atomic force microscopy [Articolo su rivista]
Raspanti, M.; Alessandrini, A.; Ottani, V.; Ruggeri, A.
abstract

Most studies on the interaction of collagen with proteoglycans, two universal components of connective tissues, use technical approaches which substantially modify the shape and size of the proteoglycans themselves. In the present study unfixed, untreated collagen fibrils from rat tail tendon were dehydrated and observed by tapping-mode atomic force microscopy. The surface of collagen fibrils immediately reveals a periodic alternation of gap and overlap zones. A thin, transverse ridge decorates the gap zone, while other filamentous structures run on the fibril surface, either parallel or perpendicular to the fibril axis. These surface structures are much enhanced by Cupromeronic Blue preincubation, while pretreatment with chondroitinase ABC removes them completely, leaving barely detectable transverse ridges. The ridge and filaments are likely to represent, respectively, the core protein and the glycosaminoglycan side chains of proteoglycans, displayed with a far better resolution than with conventional histochemical or immunohistochemical techniques. Our data suggest that proteoglycan molecules are capable of different, multiple interactions with the collagen fibril surface as well as with each other.

1996 - Collagen fibril surface: TMAFM, FEG-SEM and freeze-etching observations [Articolo su rivista]
Raspanti, M.; Alessandrini, A.; Gobbi, P.; Ruggeri, A.
abstract

Native, unfixed collagen fibrils from rat tail tendon were dehydrated following different procedures and observed under a FEG-SEM and an AFM operated in Tapping Mode (TMAFM). Freeze-etched, untreated fibrils from the same tissue were also observed for comparison. The most notable features of the fibril surface, i.e., the gap/overlap alternation and three prominent intraperiod ridges, were simultaneously visible only in freeze-etched specimens, while under the SEM and the TMAFM their appearance was dependent on both the dehydration procedure and the visualization technique. The different susceptibility of the collagen fibril surface structures to various treatments clearly implies the existence of domains of different composition. Moreover, identical specimens were imaged differently by SEM and TMAFM, highlighting instrument-specific advantages and limitations. The onset of dehydration-dependent, procedure-specific artifacts should be considered in high-resolution studies of connective tissues. As for any biological specimen, the final aspect of collagen fibrils is determined no less by the preliminary treatments than by the visualization approach.