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Bacterial infections are among the major clinical complications causing relatively high-mortality rates after implantation of medical devices. About 65% of such infections rely heavily on the formation of biofilms that are known to display remarkable resistance to traditional antibiotic therapies. This chapter provides the readers with notes on bacterial classification and structure and aims to present a general overview of the processes of bacterial adhesion on biotic and abiotic surfaces and biofilm formation, summarizing some of the most popular and up-to-date methodologies used to engineer nonfouling surfaces and materials in general. Furthermore, state-of-the-art techniques and technologies successfully applied to study bacterial adhesion and test standards used to assess the antimicrobial effectiveness of conventional materials are thoroughly described and critically discussed as well. Hints about the inadequacy of current standards to evaluate the antimicrobial effectiveness of novel materials are also given.

A number of smart nonviral gene delivery vectors relying on bioresponsiveness have been introduced in the past few years to overcome the limits of the first generation of gene carriers. Among them, redox-sensitive lipidic and polymeric vectors exploit the presence of disulfide bonds in their structure to take advantage of the highly reductive intracellular milieu and to promote complex unpacking and nucleic acids release after cellular uptake (disulfide linker strategy). Glutathione (GSH) has been often identified as the leading actor in the intracellular reduction of bioreducible vectors but their actual mechanisms of action have been rarely investigated in depth and doubts about the real effectiveness of the disulfide linker strategy have been raised. Herein, we outline a simple protocol for the preparation and investigation of nano-sized reducible cationic liposomes, focusing on their thorough characterization and optimization as gene delivery vectors. In addition, we carefully describe the techniques and procedures necessary for the assessment of the bioreducibility of the vectors and to demonstrate that the GSH-mediated intracellular cleavage of disulfide bonds is a pivotal step in their transfection process. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and of the reducible cationic lipid SS14 are reported as a practical example but the proposed protocol can be easily shifted to other formulations of reducible lipids/liposomes and to reducible polymers.

Background.Titanium and its alloy represent the commonly used material in orthopedic joint prosthetics and dental implantology fields due to their excellent mechanical properties and biocompatibility. Among the different causes of orthopaedic and dental implants failure, the event of infections still represents the most serious and devastating complication that may involve biomaterial devices. Purpose. The aim of the present work is to develop an innovative osteointegrative and antibacterial biomimetic coating and to perform the chemical-physical and in vitro biological characterization of the coating using SAOS-2 cell line. Among the objects we intend to study the antibacterial properties of the coating against both Gram-positive and Gram-negative bacteria strains. The treatment is developed using the Anodic Spark Deposition (ASD) electrochemical technique. Methods. The proposed antibacterial ASD treatment was obtained in an electrochemical solution containing silicon, calcium, phosphorous, sodium and silver nanoparticles. Surface morphology was characterized using SEM and laser profilometry. A qualitative analysis of the chemical composition of the coating was assessed by EDS. The adhesion properties of the coating to the titanium bulk were performed with a 3 points bending test. SAOS-2 osteoblastic cell line spreading and morphology were investigated using SEM and Phalloidin staining after 24h, 48h and 72h of cell culture on the different coating, while the viability was assessed using Alamar Blue assay after 1, 4 and 7 day of cell culture. The bacterial adhesion and the antibacterial properties were investigated after 3h and 24h of incubation with Streptococcus mutans, Streptococcus epidermidis and Escherichia Coli bacterial strains. Results. The proposed anodization treatment was capable to provide a chemical and morphological modified titanium oxide layer, adherent and characterized by micropourous morphology resulted enriched by calcium, silicon, phosphorous and silver. The preliminary biological characterization showed an optimal SAOS-2 cell adhesion and proliferation. A strong antibacterial effect against both Gram-positive and Gram-negative strain has been shown. Conclusions. Considering the results obtained in the present study, we believe that the novel biomimetic antibacterial treatment represents a promising treatment capable to enhance the osteointegration and contemporaneously confer antibacterial properties to titanium for dental and orthopaedic fields.

International audience; Small interfering RNAs (siRNAs) are double strand RNA fragments of short sequence ([similar]20 bp). RNA interference came into focus only 13 years ago as a major biological breakthrough and, since then, many studies have described the involvement of siRNA in gene silencing. Application to gene therapy is extremely promising, provided that appropriate vectors are used. Optimising transfection efficacy strongly relies on the knowledge and tuning of physicochemical properties of transfection complexes, such as size, surface charge and internal interactions, which govern in vitro and in vivo stability. Here we report a study on siRNA complexation with micelles of two types of divalent cationic surfactants, i.e. three Gemini bis(quaternary ammonium) bromide with variable spacer length (12-3-12, 12-6-12, 12-12-12) and one weak electrolyte surfactant with a triazine polar head. The process of complex formation was followed by SANS, DLS and zeta potential. Charge density on micelles and counterion exchange were key factors in determining the extent of complexation, as it happens to polymer electrolytes interacting with micelles. A description of complex formation was given in terms of liquid-liquid micro-phase separation, due to internally structured coacervates progressively nucleating from the micelle solution upon siRNA addition. An affinity order between surfactants and siRNA could be established on the basis of the obtained results and their comparison.

Gene therapy is emerging as a revolutionary alternative to conventional therapeutic approaches. However, its clinical application is still hampered by the lack of safe and effective gene delivery techniques. Among the plethora of diverse approaches used to ferry nucleic acids into target cells, non-viral vectors represent promising and safer alternatives to viruses and physical techniques. Both cationic lipids and polymers spontaneously wrap and shrink the genetic material in complexes named lipoplexes and polyplexes, respectively, thereby protecting it and shielding its negative charges. The development of non-viral vectors commenced more than two decades ago. Since then, some major classes of interesting molecules have been identified and modified to optimize their properties. However, the way towards the final goal of gene delivery, i.e. protein expression or gene silencing, is filled with obstacles and current non-viral carriers still have concerns about their overall efficiency. We strongly believe that the future of non-viral gene delivery relies on the development of multifunctional vectors specifically tailored with diverse functionalities that act more like viruses. Although these vectors are still a long way from clinical practice they are the ideal platform to effectively shuttle the genetic material to target cells in a safe and controlled way. In this review, after briefly introducing the basis of gene delivery and therapeutic applications we discuss the main polymeric and lipidic vectors utilized for gene delivery, focusing on the strategies adopted to overcome the major weaknesses inherent to their still limited activity, on the way towards ideal multifunctional vectors.

Purpose Since bacterial pollution is more troublesome than other nonbiologic air pollutants, the need to control airborne micro-organisms has led to renewed interest in filter media for air filtration in indoor environments. Although mechanical filtration of aerosols by HEPA systems is the most common method for particle removal, these filters characterized by high efficiency usually reveal a higher drop in pressure and noise and are very expensive. On this basis, we aimed to develop novel, very effective air filters for removal of airborne bacteria from confined environments. Methods Parallelepiped filters surrounded by a cardboard frame were manufactured by aligning strips of corrugated cardboard and were assessed in terms of airflow rate reduction. Cardboard filters were soaked in isopropanol or used untreated in in vitro experiments for assessment of their antibacterial effect against E. coli and in a testing chamber for assessment of airborne bacterial removal. The surface morphology of cardboard specimens was also investigated by Scanning Electron Microscopy (SEM). Results Cardboard filters determined a very low decrease in airflow rate. Although specimens showed no antimicrobial behavior, untreated filters showed a maximum of 77% abatement of the airborne bacteria and the alcohol treatment of filters further increased their effectiveness by 14% probably because of their more convoluted surface. Conclusions This work disclosed corrugated cardboard-based filters as promising tools for the air treatment of indoor environments because of their excellent microbial abatement properties. Moreover, cardboard is lightweight, inexpensive, and eco-friendly material, and corrugated cardboard-based air filters are very simple in shaping, mounting, and replacing existing ventilation systems.

Genomics is the study of an organism's genome aimed at the functional specification of the different parts of the sequence that comprise the blueprint of the living cell to unveil the mechanisms of the physiology of the cell and its basic, develop- mental, and tissue-specific processes. Proteomics is the comprehensive study of the executive molecules of the cell coded by the genome, further raising the level of complexity, because of the large amplification in the number, going from genes to proteins, and to the sophisticated struc- tural and functional characterization of protein products, which confer specific biochemical properties. While continuous progress in technology provides new experimental solutions to study and measure the behavior of genes and proteins in the cell, the analysis and the management of biological data cannot be uncoupled from the use of mathema- tics, statistics, and informatics disciplines that play a key role in modern molecular biology. together, genomics and proteomics, meant as complementary approaches, delineate the framework of modern molecular medicine, where the knowledge of the functional mechanisms on a subcellular scale, both under physiologic and pathologic conditions, may lead to an improvement in diagnosis, therapy, and drug development.

Vascular tissue engineering combines cells with scaffold materials in vitro aiming the development of physiologically relevant vascular models. For natural scaffolds such as collagen gels, where cells can be mixed with the material solution before gelation, cell seeding density is a key parameter that can affect extracellular matrix deposition and remodeling. Nonetheless, this parameter is often overlooked and densities sensitively lower than those of native tissues, are usually employed. Herein, the effect of seeding density on the maturation of tubular collagen gel-based scaffolds cellularized with smooth muscle cells is investigated. The compaction, the expression, and deposition of key vascular proteins and the resulting mechanical properties of the constructs are evaluated up to 1 week of maturation. Results show that increasing cell seeding density accelerates cell-mediated gel compaction, enhances elastin expression (more than sevenfold increase at the highest density, Day 7) and finally improves the overall mechanical properties of constructs. Of note, the tensile equilibrium elastic modulus, evaluated by stress-relaxation tests, reach values comparable to native arteries for the highest cell density, after a 7-day maturation. Altogether, these results show that higher cell seeding densities promote the rapid maturation of collagen gel-based vascular constructs toward structural and mechanical properties better mimicking native arteries.

Covalent attachment of a phenolic antioxidant analogue of α-tocopherol to graphite-coated magnetic cobalt nanoparticles (CoNPs) provided a novel magnetically responsive antioxidant capable of preventing the autoxidation of organic materials and showing a reduced toxicity toward human cells.

Waste from Electric and Electronic Equipment (WEEE) represent the fastest growing waste stream in Europe. The large amount of high values material (i.e. key metals) in these End of Life products, increases the industrial interests in WEEE recycling. Despite the evident economical potentialities of this sector, the high products variability and complexity make the WEEE recycling processes a challenge task, especially considering that mechanical processes currently used by recycling companies are extremely rigid and that the on-line control strategies are not integrated in such a plants. In fact, if the continuous technological evolution increases the complexity of products, requiring the integration of proper control technologies for the characterization of fine particles mixtures, on the other hand, the variability of EoL products calls for the integration of optimization strategies in order to ensure process reconfigurability. In this context, the implementation of a HyperSpectral Imaging (HSI) system can represent a suitable technology able to improve the WEEE recycling efficiency, supporting the process control, operation and reconfiguration. This work presents a detailed step-by-step description of research activities supporting the development of a HSI classification module for fine particles mixtures (lower than 2 mm) characterization of shredded EoL products. An example application of HSI system, implemented at the Demanufacturing Pilot Plant at ITIA-CNR, is presented.The application integrates both hardware and software systems to improve the process recycling rates and the output products quality, gathering in-line data about the processed mixture composition using HSI system and adjusting the parameters of the downstream electrostatic separation process according to the specific mixture under treatment. The preliminary experimental results, obtained by processing shredded Printed Circuit Boards (PCBs), show that a considerable improvement in the output recovery can be achieved by applying an intelligent mechanical separation process. These results pave the way to a next generation of highly adaptable demanufacturing systems.

A number of smart nonviral gene delivery vectors relying on bioresponsiveness have been introduced in the past few years to overcome the limits of the first generation of gene carriers. Among them, redox-sensitive lipidic and polymeric vectors exploit the presence of disulfide bonds in their structure to take advantage of the highly reductive intracellular milieu and to promote complex unpacking and nucleic acids release after cellular uptake (disulfide linker strategy). Glutathione (GSH) has been often identified as the leading actor in the intracellular reduction of bioreducible vectors but their actual mechanisms of action have been rarely investigated in depth and doubts about the real effectiveness of the disulfide linker strategy have been raised. Herein, we outline a simple protocol for the preparation and investigation of nano-sized reducible cationic liposomes, focusing on their thorough characterization and optimization as gene delivery vectors. In addition, we carefully describe the techniques and procedures necessary for the assessment of the bioreducibility of the vectors and to demonstrate that the GSH-mediated intracellular cleavage of disulfide bonds is a pivotal step in their transfection process. Liposomes composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DOPE), and of the reducible cationic lipid SS14 are reported as a practical example but the proposed protocol can be easily shifted to other formulations of reducible lipids/liposomes and to reducible polymers.

Background: Despite the use of currently optimized lipofection conditions, including transfection in serum-depleted media, the efficiency of gene transfer is low and high transfection rates often induce cytotoxicity. A lipid formulation with transfection efficiency not inhibited by serum would provide an advance towards in vivo applications. Methods: We explored the ability of the cationic lipid SH-14 to dimerize upon DNA and form lipoplexes, and potentially release nucleic acids in the intracellular reducing milieu. We investigated the critical micelle-forming concentration of SH-14 and its intrinsic toxicity, size and Zeta potential measurements, the in vitro cytotoxicity of SH-14/ plasmid DNA lipoplexes and their ability to transfect cells. Results: Among all the charge ratios (CR, +/-) tested, lipoplexes at CR 10 with a mean diameter of 295 nm and a surface charge of +20 mV, exhibited the best compromise between transfection efficiency and tolerability. SH-14 presented the same cytotoxicity level whether alone or complexed in lipoplexes. Lipofections carried out in serum-free medium shared a transfection efficiency, on average, of 40% and a cytotoxicity of 38%. An increase of 73% in transfection efficiency and 24% in cell viability were obtained, extending lipofection over 48 h in complete-medium. Moreover, when serum concentration was increased from 10% to 50%, a three-fold increase in plasmid dose led to more than 72% of cells being transfected with almost no sign of cytotoxicity. Conclusions: Overall, SH-14 presents good potential as a novel transfection reagent to be used in the presence of serum.

Hyperspectral remote sensing can support exploration activities helping in the recognition of lithologycal outcrops over extended areas. This is mainly due to the nature of the mineral reflectance spectra which feature very narrow absorption peaks, therefore requiring hyperspectral sensors to be assessed. In this work, the capabilities for geolithology recognition of aerial hyperspectral data have been tested to map outcrops in an Alpine carbonates test basin, located in the North-Eastern part of Italy. Two hyperspectral datasets, from MIVIS and SASI sensors, have been classified to retrieve the geolithological map of the test basin. The classification procedure was based on a decision tree approach, in order to combine different spectral and morphological features, in a flexible, robust and scalable solution. Despite the geological complexity of the study area (Alpine environment, spread vegetation cover, strong presence of shadow areas and presence of transitional geologic formations) the results, as compared to reference geological maps, prove satisfactory mapping capabilities, particularly for carbonatic rocks. In fact, the use of aerial hyperspectral data not only allows their accurate detection, but also the distinction between dolostones and limestones. The main classification issues are related to the outcrops' extent which, in a such lithology-fragmented area, avoids the detection of many small outcrops.

The racemic α-trifluoromethyl-α-amino-β-sulfone hydroxamates 1 were synthesized by means of a nucleophilic addition of sulfur-stabilized carbanions to a N -Cbz imine of trifluoropyruvate ( 4 ). The free amino derivative 1a was the most potent inhibitor of both MMP-3 (stromelysin-1) and MMP-9 (gelatinase-B), showing an IC 50 = 14 nM and 1 nM, respectively, and excellent selectivity versus MMP-1 (>5000-fold difference in inhibitory capacity). The N -Me derivative 1b was the most selective for MMP-3 with respect to MMP-9 (62-fold difference).

L'agricoltura di precisione (in inglese Precision Farming), si differenzia dalle pratiche agricole convenzionali, per l'utilizzo di moderni sistemi di invio e ricezione dati GPS o informazioni derivanti da sensori remoti usati come input per il controllo delle macchine agricole e delle loro componenti. Questo studio analizza la possibilità di utilizzare il telerilevamento per la produzione di mappe ed informazioni utili per la Precision Farming (De la fuente et al., 2013). Durante la stagione estiva 2014 sono state acquisite due immagini satellitari WorldView2 ad altissima risoluzione (2 m), il 16 Luglio e 17 Agosto, con l'obiettivo di testare le informazioni estratte dalle immagini come strumento predittivo della resa finale di alcuni campi di riso e per indirizzare pratiche agricole come le fertilizzazioni. Per entrambe le immagini satellitari sono stati calcolati diversi indici di vegetazione, i quali sono stati messi in relazione con i dati di resa acquisiti a fine stagione da una mietitrebbia dotata di pesa e posizionamento GPS, al fine di: 1) individuare l'indice con le migliori prestazioni e 2) valutare il corretto periodo di acquisizione. Il confronto delle correlazioni tra gli indici di vegetazione e i dati di resa finale hanno evidenziato valori massimi di correlazione R2 intorno a 0.6 per gli indici con le migliori prestazioni (NDVI, NDRE). Inoltre la possibilità di poter geo-localizzare la variabilità del vigore durante la stagione, permette di fornire indicazioni per attuare un'agricoltura di precisione e di ottenere vantaggi dal punto di vista sia economico che ambientale legati, per esempio, alla razionalizzazione nell'uso del concime (Zhang, Wang, & Wang, 2002).

The widespread use of gene therapy as a therapeutic tool relies on the development of DNA-carrying vehicles devoid of any safety concerns. In contrast to viral vectors, non-viral gene carriers show promise in this perspective, although their low transfection efficiency leads to the necessity to carry out further optimizations. In order to overcome the limitations of traditional macroscale approaches, which mainly consist of time-consuming and simplified models, a microfluidic strategy has been developed to carry out transfection studies on single cells in a high-throughput and deterministic fashion. A single cell trapping mechanism has been implemented, based on the dynamic variation of fluidic resistances. For this purpose, we designed a round-shaped culture chamber integrated with a bottom trapping junction, which modulates the hydraulic resistance. Several layouts of the chamber were designed and computationally validated for optimization of the single cell trapping efficacy. The optimized chamber layout was integrated in a polydimethylsiloxane (PDMS) microfluidic platform presenting two main functionalities: (i) 288 chambers for trapping single cells, and (ii) a serial dilution generator with chaotic mixing properties, able to deliver to the chambers both soluble factors and non-diffusive particles (i.e., polymer/DNA complexes, polyplexes) under spatio-temporally controlled chemical patterns. The devices were experimentally validated and allowed the trapping of individual human glioblastoma–astrocytoma epithelial-like cells (U87-MG) with a trapping efficacy of about 40%. The cells were cultured within the device and underwent preliminary transfection experiments using 25 kDa linear polyethylenimine (lPEI)-based polyplexes, confirming the potentiality of the proposed platform for the future high-throughput screening of gene delivery vectors and for the optimization of transfection protocols.

Cathodic polarization is a powerful technique largely used for the deposition of thin films on metallic substrates; it offers the distinctive advantage of an easy control over the composition, thickness, and morphology of the films by simply adjusting the process parameters such as the electrolyte bath composition, the applied potential (or current density), and the process duration. In this work, electrochemical deposition (ECD) was exploited to engender biopolymer blends composed by chitosan/collagen and chitosan/poly (ethylene oxide) and deposited at different weight ratios (5:1, 3:1) and compared to pristine material. Our findings demonstrate that ECD is an effective technique for the preparation of scaffolds made of chitosan blends in which morphology and mechanical properties can be optimized via scaffold composition.

The bioisosterism of the trifluoromethyl group, namely its capacity to act as a replacement for groups with similar size or shape without substantially altering key biological properties such as binding affinity, remains a controversial issue.

Satellite remote sensing is the process of collecting information about the Earth surface from the space through the measure of electromagnetic radiation. Nowadays, remote sensing is a mature technology used to extract, analyze, and detect changes of geographic and thematic information over large areas, inaccessible sites, or where only limited knowledge is available. In this chapter, we describe how satellite’s data collected over Multan (Pakistan) have been used for mapping and monitoring the dynamics of the urban area. A multi-scale approach allowed to evaluate the urban growth of the municipality area occurred in the last 2 decades with medium-resolution Landsat-5/TM time series. Urban green plots and infrastructures (buildings and roads) have been mapped at the local scale of the historic Walled City with the state-of-the-art GeoEye-1 and WorldView-2 very high-resolution multispectral imagery.

Herein we demonstrate that fluorescence lifetime measurements of DNA-dye SYBR Green I provide significant information for the physicochemical characterization of polymeric vectors/DNA complexes (polyplexes). The relationships among fluorescence amplitude/lifetime, structure and transfection efficiency of polyplexes is identified and discussed.

In questo contributo sono presentati i risultati di un progetto di ricerca che ha avuto come obiettivo la mappatura delle coperture in cemento-amianto nei Comuni di Biassono, Lissone, Monza, Muggio? e Seregno (MB) su un'area di circa 65 km2. Lo studio ha previsto l'acquisizione di immagini iperspettrali in data 5 luglio 2013 tramite il sensore aviotrasportato MIVIS (Multispectral Infrared and Visible Imaging Spectrometer) operante in 102 canali nell'intervallo spettrale compreso tra il visibile e l'infrarosso termico. Sono inoltre state effettuate misure in campo con uno spettroradiometro portatile Fieldspec FR (ASD, USA) su coperture a diverso stato di deterioramento, allo scopo di caratterizzare il loro comportamento spettrale. Le immagini sono state corrette geometricamente ed atmosfericamente e successivamente classificate in modo automatico con l'algoritmo SAM (Spectral Angle Mapper) utilizzando un training set individuato con l'aiuto di ortofoto e sulla base di informazioni sulla presenza di coperture in cemento-amianto fornite dai Comuni. Le misure a terra hanno permesso di definire un indice spettrale relazionato all'albedo delle lastre in cemento-amianto e alla presenza di muschi e licheni sulle stesse che e? stato successivamente utilizzato per discriminare, a partire dalle immagini aeree, tetti piu? deteriorati da tetti meglio conservati. Tale approccio ha permesso di suggerire ai Comuni una diversa priorita? di intervento nella loro rimozione prevista dal PRAL (Piano Regionale Amianto Lombardia) redatto nel 2005, che pone tra gli obiettivi strategici l'eliminazione dell'amianto dal territorio lombardo entro dieci anni dalla sua entrata in vigore. I dati relativi alle coperture individuate sono stati gestiti in ambiente GIS (Geographic Information System) al fine di fornire ai Comuni il riferimento catastale di ogni singola proprieta? da sottoporre a bonifica.

Satellite remote sensing is the process of collecting information about the Earth surface from the space through the measure of electromagnetic radiation. Nowadays, remote sensing is a mature technology used to extract, analyze, and detect changes of geographic and thematic information over large areas, inaccessible sites, or where only limited knowledge is available. In this chapter, we describe how satellite's data collected over Multan (Pakistan) have been used for mapping and monitoring the dynamics of the urban area. A multi-scale approach allowed to evaluate the urban growth of the municipality area occurred in the last 2 decades with medium-resolution Landsat-5/TM time series. Urban green plots and infrastructures (buildings and roads) have been mapped at the local scale of the historic Walled City with the state-of-the-art GeoEye-1 and WorldView-2 very high-resolution multispectral imagery.

Land-cover/land-use thematic maps are a major need in urban and country planning. This paper demonstrates the capabilities of Object Based Image Analysis in multi-scale thematic classification of a complex sub-urban landscape with simultaneous presence of agricultural, residential and industrial areas using pan-sharpened very high resolution satellite imagery. The classification process was carried out step by step through the creation of different hierarchical segmentation levels and exploiting spectral, geometric and relational features. The framework returned a detailed land-cover/land-use map with a Cohen's kappa coefficient of 0.84 and an overall accuracy of 85%.

Polyplexes are nanoparticles formed by the self-assembly of DNA/RNA and cationic polymers specifically designed to deliver exogenous genetic material to cells by a process called transfection. There is a general consensus that a subtle balance between sufficient extracellular protection and intracellular release of nucleic acids is a key factor for successful gene delivery. Therefore, there is a strong need to develop suitable tools and techniques for enabling the monitoring of the stability of polyplexes in the biological environment they face during transfection. In this work we propose time-resolved fluorescence spectroscopy in combination with SYBR Green I-DNA dye as a reliable tool for the in-depth characterization of the DNA/vector complexation state. As a proof of concept, we provide essential information on the assembly and disassembly of complexes formed between DNA and each of three cationic polymers, namely a novel promising chitosan-graft-branched polyethylenimine copolymer (Chi-g-bPEI), one of its building block 2 kDa bPEI and the gold standard transfectant 25 kDa bPEI. Our results highlight the higher information content provided by the time-resolved studies of SYBR Green I/DNA, as compared to conventional steady state measurements of ethidium bromide/DNA that enabled us to draw relationships among fluorescence lifetime, polyplex structural changes and transfection efficiency.

Efficient delivery of nucleic acids into cells is a promising technique to modulate cellular gene expression for therapeutic and research applications. Cationic lipid-based liposomes represent one of the most intensively studied and employed nonviral vectors. They are positively charged at physiological pH and spontaneously self-assemble with polyanionic nucleic acids forming nanoscaled complexes named lipoplexes. Here, we draft a simple protocol for the development, characterization, optimization, and screening of liposomal formulations for in vitro gene delivery. In particular, we report as a practical example a quick method to formulate and extrude nanometer-sized unilamellar cationic vesicles composed of DOTAP as cationic lipid and DOPE as zwitterionic helper lipid at 1:1 molar ratio. The physico-chemical characterization of liposomes and lipoplexes involves the measurement of mean diameter and overall surface charge using Dynamic Light Scattering (DLS) and Laser Doppler Microelectrophoresis. The outlined transfection procedure takes into account several experimental parameters affecting the in vitro performance of gene delivery systems, paying special attention to the charge ratio (CR). Gene delivery effectiveness is evaluated both in terms of transfection efficiency and cytotoxicity of the vector to find the optimal transfection conditions. Importantly, the proposed protocol can be easily shifted to different types of nonviral vectors.

Gene delivery is the science of transferring genetic material into cells by means of a vector to alter cellular function or structure at a molecular level. In this context, a number of nucleic acid-based drugs have been proposed and experimented so far and, as they act on distinct steps along the gene transcription–translation pathway, specific delivery strategies are required to elicit the desired outcome. Cationic lipids and polymers, collectively known as non-viral delivery systems, have thus made their breakthrough in basic and medical research. Albeit they are promising alternatives to viral vectors, their therapeutic application is still rather limited as high transfection efficiencies are normally associated to adverse cytotoxic side effects. In this scenario, drawing inspiration from processes naturally occurring in vivo, major strides forward have been made in the development of more effective materials for gene delivery applications. Specifically, smart vectors sensitive to a variety of physiological stimuli such as cell enzymes, redox status, and pH are substantially changing the landscape of gene delivery by helping to overcome some of the systemic and intracellular barriers that viral vectors naturally evade. Herein, after summarizing the state-of-the-art information regarding the use of nucleic acids as drugs, we review the main bottlenecks still limiting the overall effectiveness of non-viral gene delivery systems. Finally, we provide a critical outline of emerging stimuli-responsive strategies and discuss challenges still existing on the road toward conceiving more efficient and safer multifunctional vectors.

The development of multifunctional vectors for efficient and safe gene delivery is one of the major challenges for scientists working in the gene therapy field. In this context, we have designed a novel type of aminoglycoside-rich dendrimers with a defined structure based on polyamidoamine (PAMAM) in order to develop efficient, nontoxic gene delivery vehicles. Three different conjugates, i.e., PAMAM G4–neamine, −paromomycin, and −neomycin, were synthesized and characterized by nuclear magnetic resonance (NMR) and MALDI analysis. The conjugates were found to self-assemble electrostatically with plasmid DNA, and unlike neamine conjugate, each at its optimum showed increased gene delivery potency compared to PAMAM G4 dendrimer in three different cell lines, along with negligible cytotoxicity. These results all disclosed aminoglycosides as suitable functionalities for tailoring safe and efficient multifunctional gene delivery vectors.

Efficient delivery of nucleic acids into cells is a promising technique to modulate cellular gene expression for therapeutic and research applications. Cationic lipid-based liposomes represent one of the most intensively studied and employed nonviral vectors. They are positively charged at physiological pH and spontaneously self-assemble with polyanionic nucleic acids forming nanoscaled complexes named lipoplexes. Here, we draft a simple protocol for the development, characterization, optimization, and screening of liposomal formulations for in vitro gene delivery. In particular, we report as a practical example a quick method to formulate and extrude nanometer-sized unilamellar cationic vesicles composed of DOTAP as cationic lipid and DOPE as zwitterionic helper lipid at 1:1 molar ratio. The physico-chemical characterization of liposomes and lipoplexes involves the measurement of mean diameter and overall surface charge using Dynamic Light Scattering (DLS) and Laser Doppler Microelectrophoresis. The outlined transfection procedure takes into account several experimental parameters affecting the in vitro performance of gene delivery systems, paying special attention to the charge ratio (CR). Gene delivery effectiveness is evaluated both in terms of transfection efficiency and cytotoxicity of the vector to find the optimal transfection conditions. Importantly, the proposed protocol can be easily shifted to different types of nonviral vectors.

Although redox-sensitive transfectants have been considered hitherto as the Holy Grail of gene delivery because of their ability to restrict the release of nucleic acids to intracellular compartments, the reasons for their sometimes lackluster performance do not seem likewise clear. To ascertain the possible influence of extracellular soluble thiols, exofacial protein sulfhydryls (EPTs) and glutathione (GSH) on the overall efficacy of bioreducible lipoplexes, we utilized a cationic gemini surfactant - SS14 - in which the two single-chain amphiphiles are held together by a suitable redox-sensitive linkage. We herein draw a big picture whereby the interaction of bioreducible lipoplexes with cells and their internalization are tightly coupled events that ultimately do affect transfection. Specifically, we provide evidence that EPTs entail the reduction-triggered disruption of SS14 lipoplexes in plain Dulbecco's Modified Eagle Medium (DMEM), thereby resulting in a considerable waste (ca. 30%) of nucleic acids and low transgene expression. The DNA release from bioreducible lipoplexes can be blunted to ca. 16% by transfecting cells in complete medium and fully reverted by preincubating them for 1 h before delivery in the same culture supernatant (i.e. preconditioning), thus significantly increasing transfection by ca. 3-fold and 10-fold, respectively. These results lead to the proposal of the protein corona as the central mediator in shielding SS14 bioreducible lipoplexes from the action of EPTs in the early phase of delivery and provide a smart solution as to how to increase their efficacy. Besides, we pinpoint associations between intracellular GSH levels and the extent of transfection. All these issues were unique to bioreducible lipoplexes. (C) 2012 Elsevier B.V. All rights reserved.

To fulfill the requirements of the 2008/50 Directive, which allows member states and regional authorities to use a combination of measurement and modeling to monitor air pollution concentration, a key approach to be properly developed and tested is the data assimilation one. In this paper, with a focus on regional domains, a comparison between optimal interpolation and Ensemble Kalman Filter is shown, to stress pros and drawbacks of the two techniques. These approaches can be used to implement a more accurate monitoring of the long-term pollution trends on a geographical domain, through an optimal combination of all the available sources of data. The two approaches are formalized and applied for a regional domain located in Northern Italy, where the PM10 level which is often higher than EU standard limits is measured.

Purpose Indoor Air Quality (IAQ) is strictly affected by the concentration of Total suspended particulate matter (TSP). Air filtration is by far the most feasible suggestion to improve IAQ. Unfortunately, highly effective HEPA filters also have a few major weaknesses that have hindered their widespread use. There is therefore a renewed interest in developing novel, cost-effective filtration systems. We have recently reported the development of cardboard-based filters for bacterial removal that were further implemented and tested herein. Methods A parallelepiped filter manufactured by aligning strips of corrugated cardboard and surrounded by a cardboard frame was specifically designed with an internal pocket holding a partially cut antistatic pleated fabric (HP). This filter, together with its parent version (CTRL) and a commercially sourced specimen (CAF), were assessed comparatively in a longtime test to assess their effectiveness on TSP removal. Results We found that the TSP abatement efficiency (E%) of the HP filter was relatively high and invariable over the 93 days of test and the pressure drop (PD%) decrease because of filter clogging was moderate. Most important, the HP filter was the most effective if assessed in terms of overall yield (Y%) and its performance was quite constant over the entire period considered. Conclusions This work disclosed this novel class of corrugated cardboard-based filters as promising tools to ameliorate IAQ in light of their good TSP removal properties that endure over time. Moreover, cardboard is a lightweight, inexpensive, and eco-friendly material and corrugated cardboard-based air filters are very easy to shape and mount on and/or replace in existing ventilation systems

This paper describes the use of airborne hyperspectral remote sensing for mapping asbestos roofs in an orographic complex area in Northern Italy, the Aosta Valley. Using training samples collected during field surveys, thematic classification was able to detect the majority of asbestos surfaces. Considering the total amount of asbestos areas validation showed a correct detection of about 80%, while considering the number of asbestos roofs correctly detected this value decreased to 43%. This difference pointed out a clear relationship between data spatial resolution and asbestos roofs area. The study served as a first approach to an extensive use of the remote sensing technology for asbestos mapping over large areas and the encouraging results will support Public Administrations for decision making strategies and policies for their removal.

BACKGROUND: Successful non-viral gene delivery currently requires compromises to achieve useful transfection levels while minimizing toxicity. Despite high molecular weight (MW) branched polyethylenimine (bPEI) is considered the gold standard polymeric transfectant, it suffers from high cytotoxicity. Inversely, its low MW counterpart is less toxic and effective in transfection. Moreover, chitosan is a highly biocompatible and biodegradable polymer but characterized by very low transfection efficiency. In this scenario, a straightforward approach widely exploited to develop effective transfectants relies on the synthesis of chitosan-graft-low MW bPEIs (Chi-g-bPEI(x)) but, despite the vast amount of work that has been done in developing promising polymeric assemblies, the possible influence of the degree of grafting on the overall behavior of copolymers for gene delivery has been largely overlooked. METHODOLOGY/PRINCIPAL FINDINGS: With the aim of providing a comprehensive evaluation of the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of copolymeric vectors, we have synthesized seven Chi-g-bPEI(x) derivatives with a variable amount of bPEI grafts (minimum: 0.6%; maximum: 8.8%). Along the Chi-g-bPEI(x) series, the higher the degree of grafting, the greater the ζ-potential and the cytotoxicity of the resulting polyplexes. Most important, in all cell lines tested the intermediate degree of grafting of 2.7% conferred low cytotoxicity and higher transfection efficiency compared to other Chi-g-bPEI(x) copolymers. We emphasize that, in transfection experiments carried out in primary articular chondrocytes, Chi-g-bPEI(2.7%) was as effective as and less cytotoxic than the gold standard 25 kDa bPEI. CONCLUSIONS/SIGNIFICANCE: This work underlines for the first time the pivotal role of the degree of grafting in modulating the overall transfection effectiveness of Chi-g-bPEI(x) copolymers. Crucially, we have demonstrated that, along the copolymer series, the fine tuning of the degree of grafting directly affected the overall charge of polyplexes and, altogether, had a direct effect on cytotoxicity.

Purpose Despite improvements in operative environment and surgical techniques, post-operative infections remain one of the most devastating complications in total joint replacement prostheses. Several efforts have been made to modify the surface of materials in order to prevent bacterial adhesion and colonization. Here, we show a one-pot electrochemical surface modification process for co-deposition of calcium phosphate and gentamicin, with the aim of triggering specific biological responses and imparting antibacterial properties on titanium alloy prostheses. Methods Gentamicin-loaded calcium phosphate coatings were deposited on Ti specimens via cathodic polarization in an electrochemical bath containing different amounts of the antibiotic salt (1–10 mg mL–1). Coatings were evaluated in terms of chemico-physical properties, via SEM/EDX, XRD, and ICP analysis, and antibacterial activity, via agar disc diffusion test on Staphylococcus aureus 8325–4 and Staphylococcus aureus SA113. Results An effective incorporation of gentamicin was achieved without any major effect on the morphology and structure. Morphology resulted in a typical plate-like brushite structure, confirmed by chemical composition and crystallographic structure. Gentamicin-loaded coatings showed an antibacterial efficacy on both staphlococcal strains, with a dose-dependent activity. Conclusions Electrochemical technology can be advantageously exploited in order to obtain coatings for bone-contact prostheses with tailored antibacterial properties.

Since DNA is not internalized efficiently by cells, the success of gene therapy depends on the availability of carriers to efficiently deliver genetic material into target cells. Gene delivery vectors can be broadly categorized into viral and non-viral ones. Non-viral gene delivery systems are represented by cationic lipids and polymers rely on the basics of supramolecular chemistry termed “self-assembling”: at physiological pH, they are cations and spontaneously form lipoplexes (for lipids) and polyplexes (for polymers) complexing nucleic acids. In this scenario, cationic polymers are commonly used as non-viral vehicles. Their effectiveness is strongly related to key parameters including DNA binding ability and stability in different environments. Time-resolved fluorescence spectroscopy of SYBR Green I (DNA dye) was carried out to characterize cationic polymer/DNA complex (polyplex) formation dispersed in aqueous solution. Both fluorescence amplitude and lifetime proved to be very sensitive to the polymer/DNA ratio (N/P ratio, +/-).

Very high secondary pollution concentrations often occur in Northern Italy, due to the density of anthropogenic emissions and frequent stagnant meteorological conditions that characterise the area. In such a situation it is important to use Chemical Transport Models (CTMs), to properly describe phenomena bringing to pollution formation and accumulation. Meteorological models are one of the drivers of CTMs and, due to their complexity, require high computational costs to be run. Within the HPC-EUROPA (Pan-European Research Infrastructure on High Performance Computing) project, the meteorological fields over Northern Italy have been simulated using RAMS, creating a database for future air quality assessments.

Regenerative medicine is a critical frontier in biomedical and clinical research. The major progresses in the last few years were driven by a strong clinical need which could benefit from regenerative medicine outcomes for the treatment of a large number of conditions including birth defects, degenerative and neoplastic diseases, and traumatic injuries. Regenerative medicine applies the principles of engineering and life sciences to enhance the comprehension of the fundamental biological mechanisms underlying the structure-function relationships in physiologic and pathologic tissues and to accomplish alternative strategies for developing in vitro biological substitutes which are able to restore, maintain, or improve tissue, and organ function. This paper reviews selected approaches currently being investigated at Politecnico di Milano in the field of regenerative medicine. Specific tissue-oriented topics are divided in three sections according to each developmental stage: in vitro study, pre-clinical study, and clinical application. In vitro studies investigate the basic phenomena related to gene delivery, stem cell behavior, tissue regeneration, and to explore dynamic culture potentiality in different applications: cardiac and skeletal muscle, cartilage, hematopoietic system, peripheral nerve, and gene delivery. Specific fields of regenerative medicine, i.e., bone, blood vessels, and ligaments engineering have already reached the preclinical stage providing promising insights for further research towards clinical applications. The translation of the results obtained during in vitro and preclinical steps into clinical organ replacement is a very challenging issue, which can offer a valid alternative to fight morbidity, organ shortage, and ethical-social problems associated with allotransplantation as shown in the clinical case reported in this review.

We present an integrated experimental-computational mechanobiology model of chondrogenesis. The response of human articular chondrocytes to culture medium perfusion, versus perfusion associated with cyclic pressurisation, versus non-perfused culture, was compared in a pellet culture model, and multiphysic computation was used to quantify oxygen transport and flow dynamics in the various culture conditions. At 2 weeks of culture, the measured cell metabolic activity and the matrix content in collagen type II and aggrecan were greatest in the perfused+pressurised pellets. The main effects of perfusion alone, relative to static controls, were to suppress collagen type I and GAG contents, which were greatest in the non-perfused pellets. All pellets showed a peripheral layer of proliferating cells, which was thickest in the perfused pellets, and most pellets showed internal gradients in cell density and matrix composition. In perfused pellets, the computed lowest oxygen concentration was 0.075 mM (7.5% tension), the maximal oxygen flux was 477.5 nmol/m(2)/s and the maximal fluid shear stress, acting on the pellet surface, was 1.8 mPa (0.018 dyn/cm(2)). In the non-perfused pellets, the lowest oxygen concentration was 0.003 mM (0.3% tension) and the maximal oxygen flux was 102.4 nmol/m(2)/s. A local correlation was observed, between the gradients in pellet properties obtained from histology, and the oxygen fields calculated with multiphysic simulation. Our results show up-regulation of hyaline matrix protein production by human chondrocytes in response to perfusion associated with cyclic pressurisation. These results could be favourably exploited in tissue engineering applications.

Since stretching plays a key role in skeletal muscle tissue development in vivo, by making use of an innovative bioreactor and a biodegradable microfibrous scaffold (DegraPol(R)) previously developed by our group, we aimed to investigate the effect of mechanical conditioning on the development of skeletal muscle engineered constructs, obtained by seeding and culturing murine skeletal muscle cells on electrospun membranes.Following 5 days of static culture, skeletal muscle constructs were transferred into the bioreactor and further cultured for 13 days, while experiencing a stretching pattern adapted from the literature to resemble mouse development and growth. Sample withdrawal occurred at the onset of cyclic stretching and after 7 and 10 days. Myosin heavy chain (MHC) accumulation in stretched constructs (D) was evaluated by Western blot analysis and immunofluorescence staining, using statically cultured samples (S) as controls.Western blot analysis of MHC on dynamically (D) and statically (S) cultured constructs at different time points showed that, at day 10, the applied stretching pattern led to an eight-fold increase in myosin accumulation in cyclically stretched constructs (D) with respect to the corresponding static controls (S). These results were confirmed by immunofluorescence staining of total sarcomeric MHC.Since previous attempts to reproduce skeletal myogenesis in vitro mainly suffered from the difficulty of driving myoblast development into an architecturally organized array of myosin expressing myotubes, the chance of inducing MHC accumulation via mechanical conditioning represents a significant step towards the generation of a functional muscle construct for skeletal muscle tissue engineering applications.

Synthetic vectors for gene delivery offer a wide variety of functional derivatization, which can be exploited to increase targeting and transfection efficacy. In this field, redox-sensitive agents based on the thiol/disulfide (–SH/–SS–) equilibrium are a class of promising transfectants. Here the thiol group content in lipoplexes formed by a triazine-based sulfhydryl surfactant (SH14) and a plasmid (pGEFP-N1) was probed by Electron Spin Resonance (ESR) of appropriately tailored nitroxides. By modelling the time decay of ESR intensity, details on the process of lipoplex formation were obtained. It was found that the concentration of available –SH groups depended on the contact time between SH14 and DNA, suggesting that lipoplex formation entailed disulfide bridge formation among SH14 molecules. This finding represents the first experimental evidence that the –SH/–SS– equilibrium plays a role in lipoplex formation when DNA is complexed by sulfhydryl-based transfecting agents, which may have profound influence on their mechanism of action.

A new yellow pigment, designated acetosellin 1 , was isolated from the mycelium of Cercosporella acetosella as a reduced azaphilone metabolite. The structure of 1 was determined by spectroscopic investigations and chemical evidence. Compound 1 possesses a novel carbon skeleton that of a naphtopyrane derivative, linked to an extensively conjugated chain. The absolute configuration was determined by NOE experiments and CD correlations.

Different Data Assimilation techniques have been formalized and applied in the context of complex nonlinear models, to describe chemistry and physics of the atmosphere. In the literature the main approaches presented are based on a) statistical interpolation (SI) techniques, including optimal interpolation methods, residual kriging methods, regression, etc… and on b) variational methods, as well as ensemble methods such as Ensemble Kalman filters (EnKF). The aim of all these methods is to combine various data sources, to provide an optimal estimate of the spatial distribution of a particular pollutant, considering the uncertainties in the measurements as well as in the models. This paper presents the Ensemble Kalman Filter (EnKF) scheme used to assimilate ozone measurements from ground monitoring stations in the simulations performed by an air quality model system. The Data Assimilation scheme has been applied to Northern Italy. Results show that the methodology highly improves the ozone estimation.