Publications by authors named "Francesca Santoro"

45 Publications

Merging office/filter paper-based tools for pre-concentring and detecting heavy metals in drinking water.

Chem Commun (Camb) 2021 Jul;57(58):7100-7103

Department of Pharmacy, University of Naples "Federico II", Naples 80131, Italy. and BAT Center-Interuniversity Center for Studies on Bioinspired Agro-Environmental Technology, University of Napoli "Federico II", Naples 80055, Italy.

A novel miniaturized and sustainable platform exploiting two merged paper-based substrates has been applied for the programmable pre-concentration of analytes of interest and electrochemical detection of mercury traces in drinking water using printable sensor strips. This strategy represents a novel versatile possibility in merging humble materials maximizing their impacts on analytical and remediation challenges.
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http://dx.doi.org/10.1039/d1cc02481gDOI Listing
July 2021

Altered heparan sulfate metabolism during development triggers dopamine-dependent autistic-behaviours in models of lysosomal storage disorders.

Nat Commun 2021 06 9;12(1):3495. Epub 2021 Jun 9.

Telethon Institute of Genetics and Medicine, Pozzuoli, Naples, Italy.

Lysosomal storage disorders characterized by altered metabolism of heparan sulfate, including Mucopolysaccharidosis (MPS) III and MPS-II, exhibit lysosomal dysfunctions leading to neurodegeneration and dementia in children. In lysosomal storage disorders, dementia is preceded by severe and therapy-resistant autistic-like symptoms of unknown cause. Using mouse and cellular models of MPS-IIIA, we discovered that autistic-like behaviours are due to increased proliferation of mesencephalic dopamine neurons originating during embryogenesis, which is not due to lysosomal dysfunction, but to altered HS function. Hyperdopaminergia and autistic-like behaviours are corrected by the dopamine D1-like receptor antagonist SCH-23390, providing a potential alternative strategy to the D2-like antagonist haloperidol that has only minimal therapeutic effects in MPS-IIIA. These findings identify embryonic dopaminergic neurodevelopmental defects due to altered function of HS leading to autistic-like behaviours in MPS-II and MPS-IIIA and support evidence showing that altered HS-related gene function is causative of autism.
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http://dx.doi.org/10.1038/s41467-021-23903-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190083PMC
June 2021

Intracellular action potential recordings from cardiomyocytes by ultrafast pulsed laser irradiation of fuzzy graphene microelectrodes.

Sci Adv 2021 Apr 7;7(15). Epub 2021 Apr 7.

Istituto Italiano di Tecnologia, Genova 16163, Italy.

Graphene with its unique electrical properties is a promising candidate for carbon-based biosensors such as microelectrodes and field effect transistors. Recently, graphene biosensors were successfully used for extracellular recording of action potentials in electrogenic cells; however, intracellular recordings remain beyond their current capabilities because of the lack of an efficient cell poration method. Here, we present a microelectrode platform consisting of out-of-plane grown three-dimensional fuzzy graphene (3DFG) that enables recording of intracellular cardiac action potentials with high signal-to-noise ratio. We exploit the generation of hot carriers by ultrafast pulsed laser for porating the cell membrane and creating an intimate contact between the 3DFG electrodes and the intracellular domain. This approach enables us to detect the effects of drugs on the action potential shape of human-derived cardiomyocytes. The 3DFG electrodes combined with laser poration may be used for all-carbon intracellular microelectrode arrays to allow monitoring of the cellular electrophysiological state.
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http://dx.doi.org/10.1126/sciadv.abd5175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026128PMC
April 2021

Outcome Features Analysis in Intramedullary Tumors of the Cervicomedullary Junction: A Surgical Series.

J Neurol Surg A Cent Eur Neurosurg 2021 May 4;82(3):225-231. Epub 2021 Feb 4.

Department of Neurology and Psychiatry, Endovascular Neurosurgery/Interventional Neuroradiology, "Sapienza" University of Rome, Rome, Italy.

Object:  The aim of this study is to investigate the impact of surgery for different cervicomedullary lesions on symptomatic pattern expression and postoperative outcome. We focused on specific outcome features of the early and late postoperative assessments. The former relies on surgery-related transient and permanent morbidity and feasibility of radicality in eloquent areas, whereas the latter on long-term course in lower grade tumors and benign tumorlike lesions (cavernomas, etc.).

Material And Methods:  We retrospectively analyzed 28 cases of intramedullary tumors of the cervicomedullary junction surgically treated at our institution between 1990 and 2018. All cases were stratified for gender, histology, macroscopic appearance, location, surgical approach, and presence of a plane of dissection (POD). Mean follow-up was 5.6 years and it was performed via periodic magnetic resonance imaging (MRI) and functional assessments (Karnofsky Performance Scale [KPS] and modified McCormick [MC] grading system).

Results:  In all, 78.5% were low-grade tumors (or benign lesions) and 21.5% were high-grade tumors. Sixty-one percent underwent median suboccipital approach, 18% a posterolateral approach, and 21% a posterior cervical approach. Gross total resection was achieved in 54% of cases, near-total resection (>90%) in 14%, and subtotal resection (50-90%) in 32% of cases. Early postoperative morbidity was 25%, but late functional evaluation in 79% of the patients showed KPS > 70 and MC grade I; only 21% of cases showed KPS < 70 and MC grades II and III at late follow-up. Mean overall survival was 7 years in low-grade tumors or cavernomas and 11.7 months in high-grade tumors. Progression-free survival at the end of follow-up was 71% (evaluated mainly on low-grade tumors).

Conclusions:  The surgical goal should be to achieve maximal cytoreduction and minimal postoperative neurologic damage. Functional outcome is influenced by the presence of a POD, radicality, histology, preoperative status, and employment of advanced neuroimaging planning and intraoperative monitoring.
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http://dx.doi.org/10.1055/s-0040-1719080DOI Listing
May 2021

Electrophysiology Read-Out Tools for Brain-on-Chip Biotechnology.

Micromachines (Basel) 2021 Jan 24;12(2). Epub 2021 Jan 24.

Enhanced Regenerative Medicine, Fondazione Istituto Italiano di Tecnologia, Via Morego, 30 - 16163 Genova, Italy.

Brain-on-Chip (BoC) biotechnology is emerging as a promising tool for biomedical and pharmaceutical research applied to the neurosciences. At the convergence between lab-on-chip and cell biology, BoC couples in vitro three-dimensional brain-like systems to an engineered microfluidics platform designed to provide an in vivo-like extrinsic microenvironment with the aim of replicating tissue- or organ-level physiological functions. BoC therefore offers the advantage of an in vitro reproduction of brain structures that is more faithful to the native correlate than what is obtained with conventional cell culture techniques. As brain function ultimately results in the generation of electrical signals, electrophysiology techniques are paramount for studying brain activity in health and disease. However, as BoC is still in its infancy, the availability of combined BoC-electrophysiology platforms is still limited. Here, we summarize the available biological substrates for BoC, starting with a historical perspective. We then describe the available tools enabling BoC electrophysiology studies, detailing their fabrication process and technical features, along with their advantages and limitations. We discuss the current and future applications of BoC electrophysiology, also expanding to complementary approaches. We conclude with an evaluation of the potential translational applications and prospective technology developments.
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http://dx.doi.org/10.3390/mi12020124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7912435PMC
January 2021

New Frontiers for Selective Biosensing with Biomembrane-Based Organic Transistors.

ACS Nano 2020 10 14;14(10):12271-12280. Epub 2020 Oct 14.

Tissue Electronics, Istituto Italiano di Tecnologia, 80125 Naples, Italy.

Biosensing plays vital roles in multiple fields, including healthcare monitoring, drug screening, disease diagnosis, and environmental pollution control. In recent years, transistor-based devices have been considered to be valid platforms for fast, low-cost sensing of diverse analytes. Without additional functionalization, however, these devices lack selectivity; several strategies have been developed for the direct immobilization of bioreceptors on the transistor surface to improve detection capabilities. In this scenario, organic transistors have gained attention for their abilities to be coupled to biological systems and to detect biomolecules. In this Perspective, we discuss recent developments in organic-transistor-based biosensors, highlighting how their coupling with artificial membranes provides a strategy to improve sensitivity and selectivity in biosensing applications. Looking at future applications, this class of biosensors represents a breakthrough starting point for implementing multimodal high-throughput screening platforms.
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http://dx.doi.org/10.1021/acsnano.0c07053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015208PMC
October 2020

Membrane Poration Mechanisms at the Cell-Nanostructure Interface.

Adv Biosyst 2019 12 6;3(12):e1900148. Epub 2019 Nov 6.

Istituto Italiano di Tecnologia, Genoa, 16163, Italy.

3D vertical nanostructures have become one of the most significant methods for interfacing cells and the nanoscale and for accessing significant intracellular functionalities such as membrane potential. As this intracellular access can be induced by means of diverse cellular membrane poration mechanisms, it is important to investigate in detail the cell condition after membrane rupture for assessing the real effects of the poration techniques on the biological environment. Indeed, differences of the membrane dynamics and reshaping have not been observed yet when the membrane-nanostructure system is locally perturbed by, for instance, diverse membrane breakage events. In this work, new insights are provided into the membrane dynamics in case of two different poration approaches, optoacoustic- and electro-poration, both mediated by the same 3D nanostructures. The experimental results offer a detailed overview on the different poration processes in terms of electrical recordings and membrane conformation.
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http://dx.doi.org/10.1002/adbi.201900148DOI Listing
December 2019

A biohybrid synapse with neurotransmitter-mediated plasticity.

Nat Mater 2020 09 15;19(9):969-973. Epub 2020 Jun 15.

Tissue Electronics, Istituto Italiano di Tecnologia, Naples, Italy.

Brain-inspired computing paradigms have led to substantial advances in the automation of visual and linguistic tasks by emulating the distributed information processing of biological systems. The similarity between artificial neural networks (ANNs) and biological systems has inspired ANN implementation in biomedical interfaces including prosthetics and brain-machine interfaces. While promising, these implementations rely on software to run ANN algorithms. Ultimately, it is desirable to build hardware ANNs that can both directly interface with living tissue and adapt based on biofeedback. The first essential step towards biologically integrated neuromorphic systems is to achieve synaptic conditioning based on biochemical signalling activity. Here, we directly couple an organic neuromorphic device with dopaminergic cells to constitute a biohybrid synapse with neurotransmitter-mediated synaptic plasticity. By mimicking the dopamine recycling machinery of the synaptic cleft, we demonstrate both long-term conditioning and recovery of the synaptic weight, paving the way towards combining artificial neuromorphic systems with biological neural networks.
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http://dx.doi.org/10.1038/s41563-020-0703-yDOI Listing
September 2020

Silicon Nanowires for Intracellular Optical Interrogation with Subcellular Resolution.

Nano Lett 2020 02 9;20(2):1226-1232. Epub 2020 Jan 9.

Tissue Electronics, Center for Advanced Biomaterials for Healthcare , Istituto Italiano di Tecnologia , 80125 Naples , Italy.

Current techniques for intracellular electrical interrogation are limited by substrate-bound devices, technically demanding methods, or insufficient spatial resolution. In this work, we use freestanding silicon nanowires to achieve photoelectric stimulation in myofibroblasts with subcellular resolution. We demonstrate that myofibroblasts spontaneously internalize silicon nanowires and subsequently remain viable and capable of mitosis. We then show that stimulation of silicon nanowires at separate intracellular locations results in local calcium fluxes in subcellular regions. Moreover, nanowire-myofibroblast hybrids electrically couple with cardiomyocytes in coculture, and photostimulation of the nanowires increases the spontaneous activation rate in coupled cardiomyocytes. Finally, we demonstrate that this methodology can be extended to the interrogation of signaling in neuron-glia interactions using nanowire-containing oligodendrocytes.
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http://dx.doi.org/10.1021/acs.nanolett.9b04624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7513588PMC
February 2020

Dynamic Manipulation of Cell Membrane Curvature by Light-Driven Reshaping of Azopolymer.

Nano Lett 2020 01 19;20(1):577-584. Epub 2019 Dec 19.

Department of Chemistry , Stanford University , 333 Campus Drive , Stanford , California 94305 , United States.

Local curvatures on the cell membrane serve as signaling hubs that promote curvature-dependent protein interactions and modulate a variety of cellular processes including endocytosis, exocytosis, and the actin cytoskeleton. However, precisely controlling the location and the degree of membrane curvature in live cells has not been possible until recently, where studies show that nanofabricated vertical structures on a substrate can imprint their shapes on the cell membrane to induce well-defined curvatures in adherent cells. Nevertheless, the intrinsic static nature of these engineered nanostructures prevents dynamic modulation of membrane curvatures. In this work, we engineer light-responsive polymer structures whose shape can be dynamically modulated by light and thus change the induced-membrane curvatures on-demand. Specifically, we fabricate three-dimensional azobenzene-based polymer structures that change from a vertical pillar to an elongated vertical bar shape upon green light illumination. We observe that U2OS cells cultured on azopolymer nanostructures rapidly respond to the topographical change of the substrate underneath. The dynamically induced high membrane curvatures at bar ends promote local accumulation of actin fibers and actin nucleator Arp2/3 complex. The ability to dynamically manipulate the membrane curvature and analyze protein response in real-time provides a new way to study curvature-dependent processes in live cells.
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http://dx.doi.org/10.1021/acs.nanolett.9b04307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7207080PMC
January 2020

Multifunctional temozolomide-loaded lipid superparamagnetic nanovectors: dual targeting and disintegration of glioblastoma spheroids by synergic chemotherapy and hyperthermia treatment.

Nanoscale 2019 Nov 30;11(44):21227-21248. Epub 2019 Oct 30.

Istituto Italiano di Tecnologia, Smart Bio-Interfaces, Viale Rinaldo Piaggio 34, 56025 Pontedera, Italy. and Politecnico di Torino, Department of Mechanical and Aerospace Engineering, Corso Duca degli Abruzzi 24, 10129 Torino, Italy.

Aiming at finding new solutions for fighting glioblastoma multiforme, one of the most aggressive and lethal human cancer, here an in vitro validation of multifunctional nanovectors for drug delivery and hyperthermia therapy is proposed. Hybrid magnetic lipid nanoparticles have been fully characterized and tested on a multi-cellular complex model resembling the tumor microenvironment. Investigations of cancer therapy based on a physical approach (namely hyperthermia) and on a pharmaceutical approach (by exploiting the chemotherapeutic drug temozolomide) have been extensively carried out, by evaluating its antiproliferative and pro-apoptotic effects on 3D models of glioblastoma multiforme. A systematic study of transcytosis and endocytosis mechanisms has been moreover performed with multiple complimentary investigations, besides a detailed description of local temperature increments following hyperthermia application. Finally, an in-depth proteomic analysis corroborated the obtained findings, which can be summarized in the preparation of a versatile, multifunctional, and effective nanoplatform able to overcome the blood-brain barrier and to induce powerful anti-cancer effects on in vitro complex models.
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http://dx.doi.org/10.1039/c9nr07976aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6867905PMC
November 2019

Living myofibroblast-silicon composites for probing electrical coupling in cardiac systems.

Proc Natl Acad Sci U S A 2019 11 17;116(45):22531-22539. Epub 2019 Oct 17.

The James Franck Institute, The University of Chicago, Chicago, IL 60637;

Traditional bioelectronics, primarily comprised of nonliving synthetic materials, lack cellular behaviors such as adaptability and motility. This shortcoming results in mechanically invasive devices and nonnatural signal transduction across cells and tissues. Moreover, resolving heterocellular electrical communication in vivo is extremely limited due to the invasiveness of traditional interconnected electrical probes. In this paper, we present a cell-silicon hybrid that integrates native cellular behavior (e.g., gap junction formation and biosignal processing) with nongenetically enabled photosensitivity. This hybrid configuration allows interconnect-free cellular modulation with subcellular spatial resolution for bioelectric studies. Specifically, we hybridize cardiac myofibroblasts with silicon nanowires and use these engineered hybrids to synchronize the electrical activity of cardiomyocytes, studying heterocellular bioelectric coupling in vitro. Thereafter, we inject the engineered myofibroblasts into heart tissues and show their ability to seamlessly integrate into contractile tissues in vivo. Finally, we apply local photostimulation with high cell specificity to tackle a long-standing debate regarding the existence of myofibroblast-cardiomyocyte electrical coupling in vivo.
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http://dx.doi.org/10.1073/pnas.1913651116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6842627PMC
November 2019

Inkjet-printed PEDOT:PSS multi-electrode arrays for low-cost in vitro electrophysiology.

Lab Chip 2019 11 16;19(22):3776-3786. Epub 2019 Oct 16.

Tissue Electronics, Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, Naples, Italy.

Multi-electrode arrays (MEAs) have become a key element in the study of cellular phenomena in vitro. Common modern MEAs are still based on costly microfabrication techniques, making them expensive tools that researchers are pushed to reuse, compromising the reproducibility and the quality of the acquired data. There is a need to develop novel fabrication strategies, able to produce disposable devices that incorporate advanced technologies beyond the standard metal electrodes on rigid substrates. Here we present an innovative fabrication process for the production of polymer-based flexible MEAs. The device fabrication exploited inkjet printing, as this low-cost manufacturing method allows for an easy and reliable patterning of conducting polymers. Poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was used as the sole conductive element of the MEAs. The physical structure and the electrical properties of the plastic/printed MEAs (pMEAs) were characterised, showing a low impedance that is maintained also in the long term. The biocompatibility of the devices was demonstrated, and their capability to successfully establish a tight coupling with cells was proved. Furthermore, the pMEAs were used to monitor the extracellular potentials from cardiac cell cultures and to record high quality electrophysiological signals from them. Our results validate the use of pMEAs as in vitro electrophysiology platforms, pushing for the adoption of innovative fabrication techniques and the use of new materials for the production of MEAs.
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http://dx.doi.org/10.1039/c9lc00636bDOI Listing
November 2019

Membrane curvature underlies actin reorganization in response to nanoscale surface topography.

Proc Natl Acad Sci U S A 2019 11 7;116(46):23143-23151. Epub 2019 Oct 7.

Department of Chemistry, Stanford University, Stanford, CA 94305;

Surface topography profoundly influences cell adhesion, differentiation, and stem cell fate control. Numerous studies using a variety of materials demonstrate that nanoscale topographies change the intracellular organization of actin cytoskeleton and therefore a broad range of cellular dynamics in live cells. However, the underlying molecular mechanism is not well understood, leaving why actin cytoskeleton responds to topographical features unexplained and therefore preventing researchers from predicting optimal topographic features for desired cell behavior. Here we demonstrate that topography-induced membrane curvature plays a crucial role in modulating intracellular actin organization. By inducing precisely controlled membrane curvatures using engineered vertical nanostructures as topographies, we find that actin fibers form at the sites of nanostructures in a curvature-dependent manner with an upper limit for the diameter of curvature at ∼400 nm. Nanotopography-induced actin fibers are branched actin nucleated by the Arp2/3 complex and are mediated by a curvature-sensing protein FBP17. Our study reveals that the formation of nanotopography-induced actin fibers drastically reduces the amount of stress fibers and mature focal adhesions to result in the reorganization of actin cytoskeleton in the entire cell. These findings establish the membrane curvature as a key linkage between surface topography and topography-induced cell signaling and behavior.
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http://dx.doi.org/10.1073/pnas.1910166116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6859365PMC
November 2019

Three-dimensionally Patterned Scaffolds Modulate the Biointerface at the Nanoscale.

Nano Lett 2019 08 3;19(8):5118-5123. Epub 2019 Jul 3.

Center for Advanced Biomaterials for Healthcare , Istituto Italiano di Tecnologia , 80125 Naples , Italy.

The main aim of cell instructive materials is to guide in a controlled way cellular behavior by fine-tuning cell-material crosstalk. In the last decades, several efforts have been spent in elucidating the relations between material cues and cellular fate at the nanoscale and in the development of novel strategies for gaining a superior control over cellular function modulation. In this context, a particular attention has been recently paid to the role played by cellular membrane rearrangement in triggering specific molecular pathways linked to the regulation of different cellular functions. Here, we characterize the effect of linear microtopographies upon cellular behavior in three-dimensional (3D) environments, with particular focus on the relations linking cytoskeleton structuration to membrane rearrangement and internalization tuning. The performed analysis shown that, by altering the cellular adhesion processes at the micro- and nanoscale, it is possible to alter the membrane physical state and cellular internalization capability. More specifically, our findings pointed out that an increased cytoskeletal structuration influences the formation of nanoinvagination membrane process at the cell-material interface and the expression of clathrin and caveolin, two of the main proteins involved in the endocytosis regulation. Moreover, we proved that such topographies enhance the engulfment of inert polystyrene nanoparticles attached on 3D patterned surfaces. Our results could give new guidelines for the design of innovative and more efficient 3D cell culture systems usable for diagnostic, therapeutic, and tissue engineering purposes.
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http://dx.doi.org/10.1021/acs.nanolett.9b01468DOI Listing
August 2019

A nanostructure platform for live-cell manipulation of membrane curvature.

Nat Protoc 2019 06 17;14(6):1772-1802. Epub 2019 May 17.

Department of Chemistry, Stanford University, Stanford, CA, USA.

Membrane curvatures are involved in essential cellular processes, such as endocytosis and exocytosis, in which they are believed to act as microdomains for protein interactions and intracellular signaling. These membrane curvatures appear and disappear dynamically, and their locations are difficult or impossible to predict. In addition, the size of these curvatures is usually below the diffraction limit of visible light, making it impossible to resolve their values using live-cell imaging. Therefore, precise manipulation of membrane curvature is important to understanding how membrane curvature is involved in intracellular processes. Recent studies show that membrane curvatures can be induced by surface topography when cells are in direct contact with engineered substrates. Here, we present detailed procedures for using nanoscale structures to manipulate membrane curvatures and probe curvature-induced phenomena in live cells. We first describe detailed procedures for the design of nanoscale structures and their fabrication using electron-beam (E-beam) lithography. The fabrication process takes 2 d, but the resultant chips can be cleaned and reused repeatedly over the course of 2 years. Then we describe how to use these nanostructures to manipulate local membrane curvatures and probe intracellular protein responses, discussing surface coating, cell plating, and fluorescence imaging in detail. Finally, we describe a procedure to characterize the nanostructure-cell membrane interface using focused ion beam and scanning electron microscopy (FIB-SEM). Nanotopography-based methods can induce stable membrane curvatures with well-defined curvature values and locations in live cells, which enables the generation of a library of curvatures for probing curvature-related intracellular processes.
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http://dx.doi.org/10.1038/s41596-019-0161-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6716504PMC
June 2019

Geosystemics View of Earthquakes.

Entropy (Basel) 2019 Apr 18;21(4). Epub 2019 Apr 18.

Planetek Italia srl, via Massaua 12, 70132 Bari, Italy.

Earthquakes are the most energetic phenomena in the lithosphere: their study and comprehension are greatly worth doing because of the obvious importance for society. Geosystemics intends to study the Earth system as a whole, looking at the possible couplings among the different geo-layers, i.e., from the earth's interior to the above atmosphere. It uses specific universal tools to integrate different methods that can be applied to multi-parameter data, often taken on different platforms (e.g., ground, marine or satellite observations). Its main objective is to understand the particular phenomenon of interest from a holistic point of view. Central is the use of entropy, together with other physical quantities that will be introduced case by case. In this paper, we will deal with earthquakes, as final part of a long-term chain of processes involving, not only the interaction between different components of the Earth's interior but also the coupling of the solid earth with the above neutral or ionized atmosphere, and finally culminating with the main rupture along the fault of concern. Particular emphasis will be given to some Italian seismic sequences.
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http://dx.doi.org/10.3390/e21040412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7514901PMC
April 2019

Cost-effective and multifunctional acquisition system for in vitro electrophysiological investigations with multi-electrode arrays.

PLoS One 2019 25;14(3):e0214017. Epub 2019 Mar 25.

Istituto Italiano di Tecnologia, Genoa, Italy.

In vitro multi-electrode array (MEA) technology is nowadays involved in a wide range of applications beyond neuroscience, such as cardiac electrophysiology and bio-interface studies. However, the cost of commercially available acquisition systems severely limits its adoption outside specialized laboratories with high budget capabilities. Thus, the availability of low-cost methods to acquire signals from MEAs is important to allow research labs worldwide to exploit this technology for an ever-expanding pool of experiments independently from their economic possibilities. Here, we provide a comprehensive toolset to assemble a multifunctional in vitro MEA acquisition system with a total cost 80% lower than standard commercial solutions. We demonstrate the capabilities of this acquisition system by employing it to i) characterize commercial MEA devices by means of electrical impedance measurements ii) record activity from cultures of HL-1 cells extracellularly, and iii) electroporate HL-1 cells through nanostructured MEAs and record intracellular signals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0214017PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6433224PMC
December 2019

Fear-specific enhancement of tactile perception is disrupted after amygdala lesion.

J Neuropsychol 2020 03 4;14(1):165-182. Epub 2019 Feb 4.

Center for Studies and Research in Cognitive Neuroscience, University of Bologna, Italy.

Tactile perception on one's own face is enhanced when viewing a fearful face being touched - as opposed to just approached - by fingers, compared to viewing other expressions, a phenomenon known as the emotional modulation of Visual Remapping of Touch (eVRT). This effect seems to be related to a preferential activation of the somatosensory system in response to threat. To test the contribution of the amygdala to this mechanism, a group of patients with unilateral lesions to the amygdala, a control group of patients with lesions in the extra-temporal regions, and a group of healthy participants completed an eVRT paradigm. They were required to detect bilateral tactile stimulation on their own cheeks, while viewing fearful, happy, or neutral faces being touched or just approached by fingers. Healthy participants and control patients confirmed that viewing a neutral face being touched - as opposed to just approached - by fingers increases tactile detection on one's own face (i.e., the typical VRT effect) and that this effect is enhanced for fearful faces, compared to neutral and happy faces. However, in patients with amygdala lesion, although the standard VRT effect was preserved for neutral faces, this was disrupted for fearful faces. This result indicates that the preferential activation of the somatosensory cortices in response to threat relies on structural integrity of the amygdala.
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http://dx.doi.org/10.1111/jnp.12178DOI Listing
March 2020

Electron Microscopy for 3D Scaffolds-Cell Biointerface Characterization.

Adv Biosyst 2019 02 9;3(2):e1800103. Epub 2018 Oct 9.

Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, 80125, Italy.

Cell fate is largely determined by interactions that occur at the interface between cells and their surrounding microenvironment. For this reason, especially in the field of tissue-engineering, there is a growing interest in developing techniques that allow evaluating cell-material interaction at the nanoscale, particularly focusing on cell adhesion processes. While for 2D culturing systems a consolidated series of tools already satisfy this need, in 3D environments, more closely recapitulating complex in vivo structures, there is still a lack of procedures furthering the comprehension of cell-material interactions. Here, the use of scanning electron microscopy coupled with a focused ion beam (SEM/FIB) for the characterization of cell interactions with 3D scaffolds obtained by different fabrication techniques is reported for the first time. The results clearly show the capability of the developed approach to preserve and finely resolve scaffold-cell interfaces highlighting details such as plasma membrane arrangement, extracellular matrix architecture and composition, and cellular structures playing a role in cell adhesion to the surface. It is anticipated that the developed approach will be relevant for the design of efficient cell-instructive platforms in the study of cellular guidance strategies for tissue-engineering applications as well as for in vitro 3D models.
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http://dx.doi.org/10.1002/adbi.201800103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340848PMC
February 2019

Assessment of Postoperative Morphologic Retinal Changes by Optical Coherence Tomography in Recipients of an Electronic Retinal Prosthesis Implant.

JAMA Ophthalmol 2019 03;137(3):272-278

Bascom Palmer Eye Institute, Department of Ophthalmology, University of Miami Miller School of Medicine, Miami, Florida.

Importance: The postoperative retinal changes at the interface between an implant electrode array and the retina and whether these anatomic changes have an association with the patient visual performance are unknown.

Objective: To report morphologic changes in recipients of an Argus II Retinal Prosthesis.

Design, Setting, And Participants: This consecutive, noncomparative case series study included a retrospective review of the preoperative and postoperative optical coherence tomography of 33 eyes among 33 individuals who underwent Argus II Retinal Prosthesis System implantation between October 28, 2011, and June 8, 2017, at 2 different centers, by the same surgeon (S.R.). Thirteen patients received an implant at Azienda Ospedaliero Universitaria Pisana, Pisa, Italy, between October 28, 2011, and October 27, 2014, and 20 patients underwent surgery at Azienda Ospedaliera Universitaria Careggi, Florence, Italy, between December 20, 2014, and June 8, 2017. Patients were excluded if they did not reach the 6-month follow-up.

Main Outcomes And Measures: All patients were evaluated before surgery, during the first postoperative day, and at 1, 3, 6, 12, and 24 months (subsequently once a year, except for patient-related adverse events), with a comprehensive ophthalmic examination, retinal fundus photography, spectral-domain optical coherence tomography, and visual function tests to evaluate the stability or improvement of their visual performance.

Results: Of the 20 patients included in the analysis, all were of white race/ethnicity, 12 (60%) were male, and the mean (SD) age was 57.4 (11.6) years. Optical coherence tomography revealed the development of a fibrosislike hyperreflective tissue limited at the interface between the array and retina in 10 eyes (50%). In 9 of 10 patients (90%), fibrosis evolved and progressed to retinal schisis. Despite the development of the fibrosis and schisis, there was no deterioration in the patient's visual performance evaluated prospectively with visual function tests (square localization and direction of motion).

Conclusions And Relevance: Optical coherence tomography may be used to observe the retinal anatomic changes in patients with an Argus II Prothesis. This analysis revealed the development of a fibrosislike hyperreflective tissue limited at the interface between array and retina that progressed to retinal schisis but with no deterioration in the patients' visual performance.
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http://dx.doi.org/10.1001/jamaophthalmol.2018.6375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6439717PMC
March 2019

Photogenerated Electrical Fields for Biomedical Applications.

Front Bioeng Biotechnol 2018 9;6:167. Epub 2018 Nov 9.

Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, Naples, Italy.

The application of electrical engineering principles to biology represents the main issue of bioelectronics, focusing on interfacing of electronics with biological systems. In particular, it includes many applications that take advantage of the peculiar optoelectronic and mechanical properties of organic or inorganic semiconductors, from sensing of biomolecules to functional substrates for cellular growth. Among these, technologies for interacting with bioelectrical signals in living systems exploiting the electrical field of biomedical devices have attracted considerable attention. In this review, we present an overview of principal applications of phototransduction for the stimulation of electrogenic and non-electrogenic cells focusing on photovoltaic-based platforms.
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http://dx.doi.org/10.3389/fbioe.2018.00167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237932PMC
November 2018

Author Correction: Plasmonic meta-electrodes allow intracellular recordings at network level on high-density CMOS-multi-electrode arrays.

Nat Nanotechnol 2018 Oct;13(10):972

Istituto Italiano di Tecnologia, Genova, Italy.

In the version of this Article originally published, the affiliation for the author Francesca Santoro was incorrectly given; it should have been 'Center for Advanced Biomaterials for Healthcare, Istituto Italiano di Tecnologia, Napoli, Italy'. This has now been corrected in all versions of the Article.
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http://dx.doi.org/10.1038/s41565-018-0261-5DOI Listing
October 2018

Plasmonic meta-electrodes allow intracellular recordings at network level on high-density CMOS-multi-electrode arrays.

Nat Nanotechnol 2018 10 13;13(10):965-971. Epub 2018 Aug 13.

Istituto Italiano di Tecnologia, Genova, Italy.

The ability to monitor electrogenic cells accurately plays a pivotal role in neuroscience, cardiology and cell biology. Despite pioneering research and long-lasting efforts, the existing methods for intracellular recording of action potentials on the large network scale suffer limitations that prevent their widespread use. Here, we introduce the concept of a meta-electrode, a planar porous electrode that mimics the optical and biological behaviour of three-dimensional plasmonic antennas but also preserves the ability to work as an electrode. Its synergistic combination with plasmonic optoacoustic poration allows commercial complementary metal-oxide semiconductor multi-electrode arrays to record intracellular action potentials in large cellular networks. We apply this approach to measure signals from human-induced pluripotent stem cell-derived cardiac cells, rodent primary cardiomyocytes and immortalized cell types and demonstrate the possibility of non-invasively testing a variety of relevant drugs. Due to its robustness and easiness of use, we expect the method will be rapidly adopted by the scientific community and by pharmaceutical companies.
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http://dx.doi.org/10.1038/s41565-018-0222-zDOI Listing
October 2018

Cells Adhering to 3D Vertical Nanostructures: Cell Membrane Reshaping without Stable Internalization.

Nano Lett 2018 09 13;18(9):6100-6105. Epub 2018 Aug 13.

Center for Advanced Biomaterials for Healthcare , Istituto Italiano di Tecnologia , Naples 80125 , Italy.

The dynamic interface between the cellular membrane and 3D nanostructures determines biological processes and guides the design of novel biomedical devices. Despite the fact that recent advancements in the fabrication of artificial biointerfaces have yielded an enhanced understanding of this interface, there remain open questions on how the cellular membrane reacts and behaves in the presence of sharp objects on the nanoscale. Here we provide a multifaceted characterization of the cellular membrane's mechanical stability when closely interacting with high-aspect-ratio 3D vertical nanostructures, providing strong evidence that vertical nanostructures spontaneously penetrate the cellular membrane to form a steady intracellular coupling only in rare cases and under specific conditions. The cell membrane is able to conform tightly over the majority of structures with various shapes while maintaining its integrity.
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http://dx.doi.org/10.1021/acs.nanolett.8b03163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485928PMC
September 2018

Cell Membrane Disruption by Vertical Micro-/Nanopillars: Role of Membrane Bending and Traction Forces.

ACS Appl Mater Interfaces 2018 Aug 21;10(34):29107-29114. Epub 2018 Aug 21.

Istituto Italiano di Tecnologia , via Morego 30 , 16163 Genova , Italy.

Gaining access to the cell interior is fundamental for many applications, such as electrical recording and drug and biomolecular delivery. A very promising technique consists of culturing cells on micro-/nanopillars. The tight adhesion and high local deformation of cells in contact with nanostructures can promote the permeabilization of lipids at the plasma membrane, providing access to the internal compartment. However, there is still much experimental controversy regarding when and how the intracellular environment is targeted and the role of the geometry and interactions with surfaces. Consequently, we investigated, by coarse-grained molecular dynamics simulations of the cell membrane, the mechanical properties of the lipid bilayer under high strain and bending conditions. We found out that a high curvature of the lipid bilayer dramatically lowers the traction force necessary to achieve membrane rupture. Afterward, we experimentally studied the permeabilization rate of the cell membrane by pillars with comparable aspect ratios but different sharpness values at the edges. The experimental data support the simulation results: even pillars with diameters in the micron range may cause local membrane disruption when their edges are sufficiently sharp. Therefore, the permeabilization likelihood is connected to the local geometric features of the pillars rather than diameter or aspect ratio. The present study can also provide significant contributions to the design of three-dimensional biointerfaces for tissue engineering and cellular growth.
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http://dx.doi.org/10.1021/acsami.8b08218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6117743PMC
August 2018

Interfacing Cells with Vertical Nanoscale Devices: Applications and Characterization.

Annu Rev Anal Chem (Palo Alto Calif) 2018 06 23;11(1):101-126. Epub 2018 Mar 23.

Department of Chemistry, Stanford University, Stanford, California 94305, USA; email:

Measurements of the intracellular state of mammalian cells often require probes or molecules to breach the tightly regulated cell membrane. Mammalian cells have been shown to grow well on vertical nanoscale structures in vitro, going out of their way to reach and tightly wrap the structures. A great deal of research has taken advantage of this interaction to bring probes close to the interface or deliver molecules with increased efficiency or ease. In turn, techniques have been developed to characterize this interface. Here, we endeavor to survey this research with an emphasis on the interface as driven by cellular mechanisms.
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http://dx.doi.org/10.1146/annurev-anchem-061417-125705DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530470PMC
June 2018

Enhanced Cell-Chip Coupling by Rapid Femtosecond Laser Patterning of Soft PEDOT:PSS Biointerfaces.

ACS Appl Mater Interfaces 2017 Nov 2;9(45):39116-39121. Epub 2017 Nov 2.

Department of Mechanical Engineering and Institute for Complex Molecular Systems, Eindhoven University of Technology , Eindhoven 5612 AZ, The Netherlands.

Interfacing soft materials with biological systems holds considerable promise for both biosensors and recording live cells. However, the interface between cells and organic substrates is not well studied, despite its crucial role in the effectiveness of the device. Furthermore, well-known cell adhesion enhancers, such as microgrooves, have not been implemented on these surfaces. Here, we present a nanoscale characterization of the cell-substrate interface for 3D laser-patterned organic electrodes by combining electrochemical impedance spectroscopy (EIS) and scanning electron microscopy/focused ion beam (SEM/FIB). We demonstrate that introducing 3D micropatterned grooves on organic surfaces enhances the cell adhesion of electrogenic cells.
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http://dx.doi.org/10.1021/acsami.7b12308DOI Listing
November 2017

Revealing the Cell-Material Interface with Nanometer Resolution by Focused Ion Beam/Scanning Electron Microscopy.

ACS Nano 2017 08 21;11(8):8320-8328. Epub 2017 Jul 21.

Stanford Institute for Materials and Energy Sciences, SLAC National Accelerator , Menlo Park, California 94025, United States.

The interface between cells and nonbiological surfaces regulates cell attachment, chronic tissue responses, and ultimately the success of medical implants or biosensors. Clinical and laboratory studies show that topological features of the surface profoundly influence cellular responses; for example, titanium surfaces with nano- and microtopographical structures enhance osteoblast attachment and host-implant integration as compared to a smooth surface. To understand how cells and tissues respond to different topographical features, it is of critical importance to directly visualize the cell-material interface at the relevant nanometer length scale. Here, we present a method for in situ examination of the cell-to-material interface at any desired location, based on focused ion beam milling and scanning electron microscopy imaging to resolve the cell membrane-to-material interface with 10 nm resolution. By examining how cell membranes interact with topographical features such as nanoscale protrusions or invaginations, we discovered that the cell membrane readily deforms inward and wraps around protruding structures, but hardly deforms outward to contour invaginating structures. This asymmetric membrane response (inward vs outward deformation) causes the cleft width between the cell membrane and the nanostructure surface to vary by more than an order of magnitude. Our results suggest that surface topology is a crucial consideration for the development of medical implants or biosensors whose performances are strongly influenced by the cell-to-material interface. We anticipate that the method can be used to explore the direct interaction of cells/tissue with medical devices such as metal implants in the future.
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http://dx.doi.org/10.1021/acsnano.7b03494DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5806611PMC
August 2017

Nanoscale manipulation of membrane curvature for probing endocytosis in live cells.

Nat Nanotechnol 2017 08 5;12(8):750-756. Epub 2017 Jun 5.

Department of Chemistry, Stanford University, 380 Roth Way, Stanford, California 94305, USA.

Clathrin-mediated endocytosis (CME) involves nanoscale bending and inward budding of the plasma membrane, by which cells regulate both the distribution of membrane proteins and the entry of extracellular species. Extensive studies have shown that CME proteins actively modulate the plasma membrane curvature. However, the reciprocal regulation of how the plasma membrane curvature affects the activities of endocytic proteins is much less explored, despite studies suggesting that membrane curvature itself can trigger biochemical reactions. This gap in our understanding is largely due to technical challenges in precisely controlling the membrane curvature in live cells. In this work, we use patterned nanostructures to generate well-defined membrane curvatures ranging from +50 nm to -500 nm radius of curvature. We find that the positively curved membranes are CME hotspots, and that key CME proteins, clathrin and dynamin, show a strong preference towards positive membrane curvatures with a radius <200 nm. Of ten CME-related proteins we examined, all show preferences for positively curved membrane. In contrast, other membrane-associated proteins and non-CME endocytic protein caveolin1 show no such curvature preference. Therefore, nanostructured substrates constitute a novel tool for investigating curvature-dependent processes in live cells.
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http://dx.doi.org/10.1038/nnano.2017.98DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5544585PMC
August 2017
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