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Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments. ispartof: Cell Mol Life Sci vol:80 issue:4 pages:93- ispartof: location:Switzerland status: Published online

The aim of this work was to develop novel chitosan (CH) based nanoparticles (NPs) for rifampicin (RIF) delivery. RIF, a lipophilic molecule, was incorporated inside NPs as a complex with an anionic cyclodextrin, sulphobutyl-ether-β-cyclodextrin (SBE-β-CD). NPs were then prepared through the ionic gelation method by exploiting the interaction between CH and SBE-β-CD-RIF complex (CH/SBE-β-CD-RIF NPs), possibly in the presence of other crosslinkers, like carboxymethylcellulose (CH/SBE-β-CD-RIF/CMC NPs) and pentasodium tripolyphosphate (CH/SBE-β-CD-RIF/TPP NPs). NPs were then characterized for their size, ζ-potential, morphology, yield, drug loading, stability, mucoadhesion, in vitro drug release and antimicrobial activity. Results demonstrated that the functional properties of loaded NPs, like their size, ζ-potential, and stability, varied on the basis of the CH/crosslinker weight ratio. Interestingly, all the developed NPs had a round shape and were characterized by high yield values and mucoadhesive properties. Among them, NPs based on CH/SBE-β-CD-RIF and CH/SBE-β-CD-RIF/CMC have gained high drug loading, provided a sustained release of RIF and showed the best antimicrobial activity. Thus, both types of NPs may be considered as promising nanocarriers for the release of RIF.

Photodynamic therapy (PDT) represents a promising therapeutic modality for cancer. Here we used an orthogonal nanoarchitectonics approach (genetic/chemical) to engineer M13 bacteriophages as targeted vectors for efficient photodynamic killing of cancer cells. M13 was genetically refactored to display on the phage tip a peptide (SYPIPDT) able to bind the epidermal growth factor receptor (EGFR). The refactored M13(EGFR) phages demonstrated EGFR-targeted tropism and were internalized by A431 cancer cells, that overexpress EGFR. Using an orthogonal approach to the genetic display, M13(EGFR) phages were then chemically modified, conjugating hundreds of Rose Bengal (RB) photosensitizing molecules on the capsid surface, without affecting the selective recognition of the SYPIPDT peptides. Upon internalization, the M13(EGFR)-RB derivatives generated intracellularly reactive oxygen species, activated by an ultralow intensity white light irradiation. The killing activity of cancer cells is observed at picomolar concentrations of the M13(EGFR) phage.

The dysregulation of the concentration of individual circulating microRNAs or small sets of them has been recognized as a marker of disease. For example, an increase of the concentration of circulating miR-17 has been linked to lung cancer and metastatic breast cancer, while its decrease has been found in multiple sclerosis and gastric cancer. Consequently, techniques for the fast, specific and simple quantitation of microRNAs are becoming crucial enablers of early diagnosis and therapeutic follow-up. DNA based biosensors can serve this purpose, overcoming some of the drawbacks of conventional lab-based techniques. Herein, we report a cost-effective, simple and robust biosensor based on localized surface plasmon resonance and hybridization chain reaction. Immobilized gold nanoparticles are used for the detection of miR-17. Specificity of the detection was achieved by the use of hairpin surface-tethered probes and the hybridization chain reaction was used to amplify the detection signal and thus extend the dynamic range of the quantitation. Less than 1 h is needed for the entire procedure that achieved a limit of detection of about 1 pM or 50 amol/measurement, well within the reported useful range for diagnostic applications. We suggest that this technology could be a promising substitute of traditional lab-based techniques for the detection and quantification of miRNAs after these are extracted from diagnostic specimens and their analysis is thus made possible., Highlights • Over- or underexpression of microRNAs can serve as biomarkers for diseases, such as cancer. • A miRNA biosensor based on localized plasmon resonance and enzyme-free amplification is here reported. • The biosensor can quantitate specific microRNA in 1 h and can be multiplexed. • The limit of detection and specificity of the biosensor is within a diagnostically-useful range.

The aim of this work was to prepare new mucoadhesive nasal decongestant nanoparticles obtained by direct crosslinking between the cationic polymer chitosan and the anionic drug cromolyn. Different chitosan/cromolyn molar ratios were used in order to obtain nanoparticles of suitable size, encapsulation efficiency/drug loading and mucoadhesion. Moreover, the ability of the nanoparticles to deliver cromolyn into and through the nasal mucosa was evaluated. The obtained positively charged nanoparticles, sized 180–400 nm, showed interesting properties in terms of yield, mucoadhesion, encapsulation efficiency and drug loading. Release and permeation/penetration data indicated the ability of the nanoparticles to retain a high amount of cromolyn inside the mucosa, which is rich in mast cells. These findings suggest developing decongestant nanoparticles for potential treatment of allergic rhinitis.

Hydroxyl terminated poly(styrene-r-methyl methacrylate) (P(S-r-MMA)) random copolymers (RCPs), with molecular weight (Mn) spanning from 1700 to 69 000 g mol−1 and equal styrene unit content, were grafted at different temperatures onto a silicon oxide surface and subsequently used to study the orientation of nanodomains with respect to the substrate, in cylinder forming polystyrene-b-poly(methyl methacrylate) (PS-b-PMMA) block copolymer thin films. When the grafting temperature increases from 250 to 310 °C, a substantial increase in the grafting rate is observed. In addition, an increase in the surface neutralization efficiency occurs thus resulting in an increase in the robustness of the surface neutralization step. These data revealed that the neutralization of the substrate is the result of a complex interplay between RCP film characteristics and underlying substrate properties that can be finely tuned by properly adjusting the temperature of the grafting process. © 2016 Society of Chemical Industry

Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3β and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC values toward GSK-3β, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 μM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, nontoxic, dual-acting compounds hitting tau cascade at two different hubs., This work was supported by the University of Bologna (RFO2015_2016), European Cooperation of Science and Technology (EUCOST) Action CA15135, Ministerio de Economia y Competitividad (CTQ2015-66313-R), Italian Ministry of Health (GR-2013-02355724), MJFF, ALZ, Alzheimer’s Research UK and the Weston Brain Institute (BAND2015). The authors thank E. De Cecco (PhD candidate, SISSA) for providing K18 and 2N4R protein samples for the in vitro assays.

DNA biosensors could overcome some of the common drawbacks of lab-based techniques for nucleic acids detection for diagnostics purposes. One of the main impediments for such applications of DNA biosensors is their lack of sensitivity: this can prevent their full exploitation in the diagnostic analytical field. DNA nanotechnology could enhance DNA biosensors and let them perform at the required high sensitivity. Well-designed, programmable self-assembly reactions can be triggered by a specific nucleic acid target. The Hybridization Chain Reaction (HCR) is a self-assembly strategy in which the target nucleic acid sequence triggers the formation of long nicked double-stranded DNA nanostructures. This can be performed in solution or on a surface, and the process can be coupled to different signal transduction schemes. We here describe the methods to design and test HCR reactions for the detection of different nucleic acid targets in solution and the procedures to exploit this strategy on surfaces with an electrochemical biosensing platform.

In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, or cell-cycle stage, make genetically-identical cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. Self-assembled DNA nanostructures can be introduced in live cells, even without the aid of transfecting agents or by altering the membrane permeability otherwise. Recently, we have worked on the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with fluorescence microscopy [3]. We have designed two types of sensitive DNA nanostructures (nanobiosensors). These are variation on the DNA tetrahedron motif introduced by the Turberfield group. Sensing of the desired target leads to a significant conformational change in the nanostructure that can be visualized (in solution or in the cells) via the spectroscopic properties of a FRET pair. For example, by exploiting and tuning the properties of an intramolecular CT-motif triple helix, we designed and tested a nanostructure that can undergo a conformational transition driven by a change in pH, in a physiologically-relevant range. We showed the functioning of such functional nanostructures and their internalization in live cultured cells.

DNA methylation catalyzed by DNA methyl transferase (MTase) is a significant epigenetic process for modulating gene expression. Abnormal levels of DNA MTase enzyme have been regarded as a cancer biomarker or a sign of bacterial diseases. We developed a novel colorimetric method to assay M.SssI MTase activity employing peroxidase-like activity of DNA template Ag/Pt NCs without using restriction enzymes. Based on inhibiting the peroxidase reaction that occurred in the TMB-H2O2 system, in the presence of MTase, a highly sensitive and selective colorimetric biosensor was fabricated with a detection limit (LOD) of 0.05 U/mL and a linear range from 0.5 to 10 U/mL. The changes in absorption intensity were monitored to quantify the M.SssI activity. This strategy had a high selectivity over other proteins. Furthermore, it is also demonstrated that this method can be used for the evaluation and screening of inhibitors for DNA MTase.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Mutations in the gene encoding copper/zinc superoxide dismutase-1 (SOD1) are responsible for most familiar cases, but the role of mutant SOD1 protein dysfunction in non-cell autonomous neurodegeneration, especially in relation to microglial activation, is still unclear. Here, we focused our study on microglial cells, which release SOD1 also through exosomes. We observed that in rat primary microglia the overexpression of the most-common SOD1 mutations linked to fALS (G93A and A4V) leads to SOD1 intracellular accumulation, which correlates to autophagy dysfunction and microglial activation. In primary contact co-cultures, fALS mutant SOD1 overexpression by microglial cells appears to be neurotoxic by itself. Treatment with the autophagy-inducer trehalose reduced mutant SOD1 accumulation in microglial cells, decreased microglial activation and abrogated neurotoxicity in the co-culture model. These data suggest that i) the alteration of the autophagic pathway due to mutant SOD1 overexpression is involved in microglial activation and neurotoxicity; ii) the induction of autophagy with trehalose reduces microglial SOD1 accumulation through proteasome degradation and activation, leading to neuroprotection. Our results provide a novel contribution towards better understanding key cellular mechanisms in non-cell autonomous ALS neurodegeneration.

DNA biosensors could overcome some of the common drawbacks of lab-based techniques for nucleic acids detection for diagnostics purposes. One of the main impediments for such applications of DNA biosensors is their lack of sensitivity: this can prevent their full exploitation in the diagnostic analytical field. DNA nanotechnology could enhance DNA biosensors and let them perform at the required high sensitivity. Well-designed, programmable self-assembly reactions can be triggered by a specific nucleic acid target. The Hybridization Chain Reaction (HCR) is a self-assembly strategy in which the target nucleic acid sequence triggers the formation of long nicked double-stranded DNA nanostructures. This can be performed in solution or on a surface, and the process can be coupled to different signal transduction schemes. We here describe the methods to design and test HCR reactions for the detection of different nucleic acid targets in solution and the procedures to exploit this strategy on surfaces with an electrochemical biosensing platform.

New low molar mass (M-n similar to 20 kg mol(-1)) polystyrene-b-poly(methyl methacrylate-co-perfluorohexylethyl acrylate) (PS-b-P(MMA-co-FA)) block copolymers were synthesized all consisting of similar to 70 mol% PS block and similar to 30 mol % P(MMA-co-FA) block. The amount of FA counits in the latter block was increased (0.5, 1, 3 and 4 mol%) in order to enhance the incompatibility between the two blocks and to form different self-assembled nanostructures. The block copolymers were thermally annealed by Rapid Thermal Processing (RTP) over wide ranges of temperatures and times. Complementary SEM and AFM analyses evidenced that the introduction of FA counits above a critical content (similar to 2 mol%) drove a self-assembly process through mixed morphologies. These were formed at a distance from the polymer-air interface and consisted of perpendicular cylinders of P(MMA-co-FA) in a PS matrix laying at the substrate- polymer interface, and surmounted PS stripes and PS dots. Modification by low amounts of FA produced block copolymer samples that self-assembled in new, mixed nanostructures, even though they possessed much lower molar masses than those of analogous well established PS-b-PMMA block copolymers.

In this study, composite nanofibrous scaffolds were obtained by electrospinning a trifluoroacetic acid solution containing B. mori silk fibroin (SF) and poly(L-lactic acid) (PLLA) in a 1:1 weight ratio. SF, PLLA and SF/PLLA nanofibres were prepared with average diameter sizes of 360±90nm, 470±240nm and 580±220nm, respectively, as assessed by SEM analysis. Vibrational and thermal analyses showed that upon blending in the SF/PLLA nanofibres, the crystallisation of PLLA was hindered by the presence of SF, which crystallized preferentially and underwent conformational changes that did not significantly change its prevailing β-sheet structure. The two components were thermodynamically compatible and the intermolecular interactions between them were revealed for the first time. Human keratinocytes were cultured on nanofibres and their viability and proliferation were determined. Preliminary in vitro tests showed that the incorporation of SF into the PLLA component enhanced cell adhesion and proliferation with respect to the unfunctionalised material. SF has been successfully used to modify the biomaterial properties and confirmed to be an efficient bioactive protein to mediate cell-biomaterial interaction.

The aim of this study was to develop chitosan-based nanoparticles that could encapsulate lipophilic molecules and deliver them to cancer cells. Nanoparticles were prepared with different molar ratios of chitosan, hyaluronic acid and sulphobutyl-ether-?-cyclodextrin and with or without curcumin. The nanosystems were characterized in terms of their size, zeta potential, morphology, encapsulation efficiency and stability in different media. Intestinal epithelial and colorectal cancer cells were treated with unloaded nanoparticles in order to study their effect on cellular membrane organization and ROS production. Finally, in vitro assays on both cellular lines were performed in order to evaluate the ability of nanoparticles to promote curcumin internalization and to study their effect on cell proliferation and cell cycle. Results show that nanoparticles were positively charged and their size increased with the increasing amounts of the anionic excipient. Nanoparticles showed good encapsulation efficiency and stability in water. Unloaded nanoparticles led to a change in lipid organization in the cellular membrane of both cell lines, without inducing ROS generation. Confocal microscopy, cell proliferation and cell cycle studies allowed the selection of the best formulation to limit curcumin cytotoxicity in normal intestinal epithelial cells and to reduce cancer cell proliferation. The latter was the result of the increase of expression for genes involved in apoptosis.

The peculiar thermal behavior of four PTFE/PMMA (Polymethylmethacrylate) core–shell nanoparticle samples, marked DV2M1, DV2M2, DV2M4, and DV2M6, was studied by combined differential scanning calorimetry and thermogravimetric analysis. The melting process of the PTFE in the various samples, subjected to annealing and thermal treatments, does not change. In contrast, a complex fractionated crystallization-type behavior for the PTFE component was observed. The nanocomposite produced by the PMMA shell fluidification features a perfect dispersion of the nanometric PTFE cores. In these conditions, only one crystallization exotherm at very high undercooling is observed, possibly deriving from the homogeneous nucleation mechanism. In contrast, when high temperature thermal treatments cause the decomposition with partial loss of the PMMA shell and allows some cores to get in contact and merge, a crystallization process structured into several components is observed. This behavior indicates that different nucleation mechanisms are active, possibly involving the participation of distinct types of active nuclei with distinct crystallization efficiencies. Finally, when the PMMA shell amount is substantially reduced by the thermal degradation, only the expected crystallization process at moderate undercooling (310 °C) is observed, corresponding to the bulk crystallization induced by the most efficient heterogeneous nuclei. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 548–554, 2010

Polytetrafluoroethylene (PTFE) latices with spherical and rod-like particles in the submicrometer size range, were employed as seeds in the emulsifier-free methylmethacrylate (MMA) emulsion polymerization to obtain PTFE-polymethylmethacrylate (PMMA) core-shell nanoparticles. Stable latices were generally obtained. No residual PTFE was found at the end of the reaction. By appropriately choosing the ratio between MMA and PTFE in the reaction mixture, particles with predetermined size and monodisperse or narrow size distribution were prepared. The high structural regularity of the core-shell samples allows the preparation of film with a periodic distribution of the cores thus ultimately leading to a well structured 2D colloidal crystal. A very peculiar crystallization behavior was observed because of the PTFE compartmentalization in the composite. (c) 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 2928-2937, 2009

A PTFE latex, with particles in the submicrometer size range, was employed as seed in the emulsifier-free emulsion copolymerization of two mixtures of acrylic and methacrylic comonomers to obtain PTFE-based core-soft shell nanoparticles. Stable latexes were obtained. By appropriately choosing the ratio between the comonomers and the PTFE seed in the reaction mixture, it was possible to obtain particles with various sizes and a narrow size distribution. The shell is swollen in water due to the presence of the ionic methacrylic acid units. A fractionated-type crystallization phenomenon of the PTFE component was observed and is ascribed to the small size of the PTFE material. The low glass transition temperatures of the shell forming materials could permit the preparation of soft matrix nanostructured films by latex deposition and water evaporation.

An increasing number of inter- and intramolecular interactions can nowadays be probed using single-molecule manipulation techniques. Protein unfolding and refolding is the most representative--though complex--of these interactions. Herein, we review the main modes of performing a force unfolding experiment: the velocity clamp and the new force clamp mode. We also compare some of the physical aspects behind the two most frequently used single-molecule manipulation instrumentations: optical tweezers and atomic force microscopes.

Nowadays, the development of experimental procedures for the determination of the secondary structure of RNA molecules is taking advantage of the novel single-molecule probing and imaging techniques. We report a method for the mapping of the secondary structure of RNA molecules spread on a flat surface by means of the atomic force microscope. Globular domains comprising groups of RNA secondary and tertiary structure elements separated by unstructured domains can be discerned in the micrographs and their position along the molecule contour can be measured directly on unstained specimens. We have analyzed the morphology of a population of single molecules of 3' fragments of the Turnip Yellow Mosaic Virus RNA shorter than 1 kb in different temperature and electrolytic conditions. We found a satisfying agreement of the shape of the imaged structures with previously available evidence. The method we have developed can be used to map also different types of RNA molecules and has the advantage of showing the distribution of the single molecule conformations within the population.

Two strategies are envisioned to improve the thermal stability of the grafted layer and to allow the processing of the random copolymer/block copolymer (RCP/BCP) system at high temperature. From one side, a high-temperature thermal treatment of a commercial a-hydroxyl omega-2,2,6,6-tetramethylpiperidinyloxy functional RCP, namely, TR58, leads to the formation of a stabilized layer able to induce the perpendicular orientation of a symmetric BCP to temperatures higher than 310 degrees C. On the other side, an alpha-hydroxyl omega-Br functional RCP, namely, BrR58, with the same molar mass and composition of TR58, was prepared by activator regenerated by electron transfer atom transfer radical polymerization. The resulting brush layer can sustain the self-assembly of the symmetric BCP for processing temperatures as high as 330 degrees C. In both systems, the disruption of the BCP film, deposited on the grafted RCP layer, occurs because of the formation of bubbles, due to a low-temperature evolution of monomers from the RCP layer. The extent of the low-temperature monomer evolution is higher for TR58 than it is for BrR58 and starts at lower temperatures. For both copolymers, the thermal treatment offsets the low-temperature monomer evolution while still maintaining surface characteristics suitable to induce the perpendicular orientation of the BCPs, thus ultimately extending the range of processing temperatures of the BCP film and consequently speeding the self-organization process.

The ordering process of asymmetric PS-b-PMMA block copolymers (BCPs) is investigated on flat SiO2 surfaces and on topographically patterned substrates. The topographic patterns consist of periodic gratings of 10 trenches defined by conventional top-down approaches and subsequently neutralized using a P(S-r-MIvIA) random copolymer (RCP). When the ordering process is accomplished on a flat surface at a temperature ranging between 180 and 230 degrees C, cylindrical microdomains perpendicularly oriented with respect to the substrate are observed irrespective of annealing temperature. In contrast, when the ordering process occurs on topographically patterned substrates, different phenomena have to be considered. The simultaneous effect of the flow around the gratings and the BCP flux from the zone located between adjacent trenches (mesa) into the inner part of the trenches results in significant thickness variations of the confined BCP film. Therefore, the amount of BCP inside the trenches depends on the width of the mesa region, which acts as a BCP reservoir. Moreover, within each trench group, the BCP thickness progressively decreases from the external to the central trenches composing the periodic grating. The thickness variation of the BCP film within the trenches strongly affects the ordering process, ultimately leading to different orientations of the microdomains within the trenches. In particular, when the annealing temperature is 190 degrees C a precise confinement of the BCP within the trenches featuring a perpendicular cylinder morphology is observed. At higher temperatures, mixed or parallel orientations of the microdomains are obtained depending on the width of the trenches in the periodic grating.

Most of the scanning force microscopy (SFM) images of supercoiled DNA on untreated mica thus far reported have not shown tight plectonemic structure seen by electron microscopy, but instead less coiled molecules and sometimes a partly "condensed" state with intimate chain-chain interactions. By observing time-lapse images of conformational changes of DNA induced by decreasing ionic strength of imaging buffer in solution SFM, we could show that the process of water rinsing, an indispensable step for preparation of dried samples, may be responsible for some of the conformational anomalies in the images previously reported. We have studied several protocols to observe supercoiled DNA molecules by SFM and discuss the merits and the demerits. Images obtained following uranyl acetate treatment may be ideal for the detection of DNA damage, as the supercoiled and nicked forms are easily distinguishable.

Cationic block copolymers, consisting of a poly(ethylene glycol) block and a block deriving from the poly(dimethylamino)ethyl methacrylate were prepared via a two-step procedure, based on the use of macroinitiators. By appropriately changing the experimental conditions and reacting the poly(dimethylamino)ethyl methacrylate block with iodo- or bromo-alkyl derivatives, a variety of ionic block copolymers with tuned physicochemical properties were prepared. These block copolymers are able to spontaneously self-assemble with plasmid DNA to produce oriented and shielded vectors, with physicochemical properties appropriate for in vivo applications. In addition, the formation of a complex between the cationic block copolymer and the plasmid DNA results in a nuclease resistance increase due to the stable nature of the complex.

Two alternative pretreatment methods for depositing metal nanoparticles on mica for atomic force microscopy (AFM) imaging are presented. The treated substrates are flat and clean, thus they are amenable of use to characterize very small nanoparticles. The methods do not require any instrumentation or particular expertise. As they are also very quick, the need for storage of the prepared substrates is avoided altogether. These proposed methods, which are compared with the results of transmission electron microscopy analysis, allow the quick sizing and characterization of nanoparticles with the atomic force microscope and could thus help expanding the user community of nanoparticle researchers who could use the AFM for their characterization needs. Microsc. Res. Tech., 2008. © 2008 Wiley-Liss, Inc.

Polynucleotide: adenosine glycosidases (PNAG) are a class of plant and bacterial enzymes commonly known as ribosome-inactivating proteins (RIP). They are presently classified as rRNA N-glycosidases in the enzyme nomenclature [EC 3.2.2.22]. Several activities on nucleic acids, other than depurination, have been attributed to PNAG: in particular modifications induced in circular plasmids, including linearisation and topological changes, and cleavage of guanidinic residues. Here we describe a chromatographic procedure to obtain nuclease-free PNAG by dye-chromatography onto Procion Red derivatized Sepharose((R)). Highly purified enzymes depurinate extensively pBR322 circular, supercoiled DNA at neutral pH and exhibit neither DNase nor DNA glycolyase activities, do not cause topological changes, and adenine is the only base released from DNA and rRNA, even at very high enzyme concentrations. A scanning force microscopy (SFM) study of pBR322 treated with saporin-S6 confirmed that (i) this PNAG binds extensively to the plasmid, (ii) the distribution of the bound saporin-S6 molecules along the DNA chain is markedly variable, (iii) plasmids already digested with saporin-S6 do not appear fragmented or topologically modified. The observations here described demonstrate that polynucleotide:adenosine glycosidase is the sole enzymatic activity of the four ribosome-inactivating proteins gelonin, momordin I, pokeweed antiviral protein from seeds and saporin-S6. These proteins belong to different families, suggesting that the findings here described may be generalized to all PNAG.

In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, cell cycle stage, make the cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. In this paper, we describe the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with epifluorescence microscopy. We report that HeLa cells internalize these nanostructures spontaneously with a higher efficiency with respect to single-stranded or double-stranded oligonucleotides. Our findings suggest that DNA tetrahedra could serve as a platform for the realization of a series of multifunctional intracellular biosensors for the analysis of single live cells. © 2014 Elsevier Inc.

The use of the scanning force microscope (SFM) to visualize and analyze chromatin fiber structures is presented. Protocols to prepare chromatin fibers for SFM imaging of fibers in air and in buffer are first discussed. Next, the conditions for acquiring high-quality SFM images such as optimal instrumental parameters, appropriate deposition substrates, and adequate procedures of sample deposition are described. It is shown that analysis and quantitation of the SFM images support an irregular, three-dimensional arrangement of nucleosomes in the native chromatin fiber. This structure is lost in linker histone-depleted fibers, which show, instead, a beads-on-a-string structure. Molecular modeling of the chromatin fiber structures and computer simulation of the SFM imaging process indicate that the natural variability of the linker length may be the major determinant of the structural irregularity of the native chromatin fiber. Removal of linker histones (H1/H5) may change the amount of DNA wrapped around the histone octamer, which in turn may induce the transition from a three-dimensional irregular helix to an extended beads-on-a-string structure. Studies of trinucleosomes indicate that both the average successive nucleosome center-to-center distance and the average angle between two successive linkers increase upon the removal of linker histone.

Contents: Overview of European regulation and standards on microbiological water analysis Risk analysis of bio-terroristic attacks on drinking water systems Sample collection procedures for Online Contaminant Monitoring System A device to extract highly diluted specimens out of large volumes of water for analysis in lab-on-a-chip detection systems Sustainable DNA/RNA release methods for in-line waterborne pathogen screening devices The microsystem based core of the DINAMICS water testing system: Design considerations and realization of the chip units Electrochemical biosensor strategies for pathogen detection in water security Biochemical and Nanotechnological Strategies for Signal Enhancement in the Detection of Nucleic Acids with biosensors Computational modelling of aqueous environments in micro and nanochannels Computational recipes of transport phenomena in micro and nanofluidics Multi-detection of waterborne pathogens in raw and treated water samples by using ultrafiltration concentration and DNA array technology Detection and enumeration of waterborne mycobacteria New molecular technologies for the rapid detection of Legionella in water Detection of virus in the water environment Design of PCR primers for the detection of waterborne bacteria Fluid structure and boundary slippage in nanoscale liquid films Understanding slip at the nanoscale in fluid flows using atomistic simulations

Early detection of protein aggregation is of great importance in the field of neurodegenerative diseases. The successful detection of the aggregation of the proteinα-synuclein in a quantitative, label-free manner by functionalising a microcantilever withα-synuclein monomers and operating it in dynamic mode in the presence ofα-synuclein monomers in solution is reported. A total mass of 6 ng ofα-synuclein was detected over 9 hours on the surface of the cantilever. The result is compared to conventional fluorescence measurements ofα-synuclein aggregation under similar conditions. It is found that the label-free cantilever detection method requires a concentration of protein 50 times smaller than that of the current method and indicated potential for significantly faster response times.

Nanosized PTFE-based core-shell particles can be prepared by emulsifier-free seed emulsion polymerization technique starting from spherical or rod-like PTFE seeds of different size. The shell can be constituted by the relatively high Tg polystyrene and polymethylmethacrylate as well as by low Tg polyacrylic copolymers. Peculiar thermal behavior of the PTFE component is observed due to the high degree of PTFE compartmentalization. A very precise control over the particle size can be exerted by properly adjusting the ratio between the monomers and the PTFE seed. Samples with uniformity ratios suited to build 2D and 3D colloidal crystals are easily prepared. 2D colloidal crystal of spheres leads to very small 2D nanostructuration, useful for the preparation of masks with a combination of nanosphere lithography and reactive ion etching. 3D colloidal crystals were also obtained featuring excellent opal quality.

PTFE/PES composites were prepared by precipitation of Radel A® into a PTFE latex containing nanoparticles with average diameters of 48 nm and spherical shape. Several samples were prepared by varying the relative ratio between the Radel A® and PTFE content. The combination of SEM and AFM analysis indicates that the precipitation of Radel A in the presence of PTFE leads mainly, if not exclusively, to a bimodal mixture of the two homoparticles. The fractionated crystallization behaviour of these samples is revealing of the PTFE dispersion degree within the Radel A® matrix. When the PTFE amount is lower than 2%, a perfect PTFE nanoparticle dispersion is obtained. When the amount of PTFE is comprised between 5 and 30%, larger PTFE clusters are obtained that, after melting, coalesce and crystallize at higher temperatures depending on the crystallization propensity of their individual heterogeneous nuclei. Finally, in case of samples 40%, only one crystallization exotherm is observed at 310 °C indicating the formation of very large clusters that after melting coalesce into wide domains. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

INTRODUCTION: Single-molecule sensitivity can be nowadays achieved when trying to characterize minute amounts of macromolecules n in a research laboratory setting. The sensitivity is significantly worse when working in the field and trying to assess the presence of a pollutant or a pathogen as quickly as possible. Point-of-care testing is the realm of biosensors and the trend of research is towards simplicity of use, low cost, reliability and, last but not least, sensitivity. In the detection of nucleic acids, the hybridization of an oligonucleotide probe with its poly-nucleotidic target is monitored by changes in physico-chemical properties of an interface, commonly induced by the use of a specific label. The probe, often bound to an oligonucleotide itself, can be a fluorescent dye, an enzyme, an electroactive moiety or other functional element. Such probes impart specificity and sensitivity to the assay, but make it drift far from simplicity of use (and low cost). METHODS AND RESULTS: Recently, label-free techniques are developing in which the presence of the analyte macromolecule itself can induce detectable physico-chemical changes. The struggle is to make such techniques sufficiently sensitive and specific. In our work, we are trying to implement DNA-based surface-bound amplification strategies that can serve to amplify the read-out signal of DNA hybridization in a label-free biosensor, such as those measuring the electrochemical properties of an electrode interface. The ‘hybridization chain reaction’ [1] is an isothermal enzyme-free strategy to trigger polymerization of oligonucleotides into a long double-stranded DNA. We have demonstrated that the ‘hybridization chain reaction’ can be also implemented (making use of available software tools [2]) in a surface-bound configuration, leading to the self-assembly of many copies of oligonucleotides on a target DNA bound on an oligonucleotide self-assembled monolayer. In another attempt, we have shown that terminal transferase (a template-free DNA polymerase) can be used to build a long polynucleotide out of the target DNA that is bound to its immobilized oligonucleotide probe. The Rolling Circle Amplification reaction is a very promising strategy to produce large amounts of DNA without the need of thermal cycling or of an accurate control of temperature. Preliminary evaluations on the Primer-Generation RCA [3] show its interest as an additional strategy to increase the amount of target DNA response from a biosensor surface. DISCUSSION & CONCLUSIONS: All these strategies will lead to the accumulation of nucleic acids at the solid-liquid interface when triggered by the probe-target recognition. This produces an amplification of the signal of a label-free biosensor. Even though the amplification factors of these implementations are still as low as 10, their further development is still possible, with the hope of avoiding or reducing the need of polymerase chain reaction in the detection of low concentrations of nucleic acids. REFERENCES: 1Dirks, R. M. and N. A. Pierce (2004). "Triggered amplification by hybridization chain reaction." Proc Natl Acad Sci U S A, 101(43): 15275-8. 2Goodman, P. (2005). "NANEV: a program employing evolutionary methods for the design of nucleic acid nanostructures" BioTechniques, 38(4): 548–550. 3Murakami, Sumaoka and Komiyama (2009) "Sensitive isothermal detection of nucleic-acid sequence by primer generation–rolling circle amplification" Nucleic Acids Research, 2009, 37, e19. ACKNOWLEDGEMENTS: This work was supported by Framework Programme 6 Integrated Project DINAMICS.

The thermal and dynamic-mechanical characteristics of three PTFE/PMMA nanoparticle samples are described. The shell forming PMMA, once isolated from the PTFE cores, exhibits a lower thermal stability than the PMMA component in the corresponding nanocomposite under both thermal and oxidative degradation conditions thus indicating a definite, though moderate, thermal reinforcement due to the morphology of the nanocomposites. An increase in the thermal stability under nitrogen atmosphere was observed as the PTFE amount increases. However under air, no difference is observed in the various systems. These observations suggest that only a physical shield can be exerted by the PTFE cores to the PMMA matrix possibly due to a weak interface between PTFE and the PMMA. This hypothesis is also substantiated by the DMA analysis.

We describe the realization of an atomic force microscope architecture designed to perform customizable experiments in a flexible and automatic way. Novel technological contributions are given by the software implementation platform (RTAI-LINUX), which is free and open source, and from a functional point of view, by the implementation of hard real-time control algorithms. Some other technical solutions such as a new way to estimate the optical lever constant are described as well. The adoption of this architecture provides many degrees of freedom in the device behavior and, furthermore, allows one to obtain a flexible experimental instrument at a relatively low cost. In particular, we show how such a system has been employed to obtain measures in sophisticated single-molecule force spectroscopy experiments [Fernandez and Li, Science 303, 1674 (2004)]. Experimental results on proteins already studied using the same methodologies are provided in order to show the reliability of the measure system. © 2009 American Institute of Physics.