Effects of cytosine methylation on DNA morphology: An atomic force microscopy study.

Methylation is one of the most important epigenetic mechanisms in eukaryotes. As a consequence of cytosine methylation, the binding of proteins that are implicated in transcription to gene promoters is severely hindered, which results in gene regulation and, eventually, gene silencing. To date, the mechanisms by which methylation biases the binding affinities of proteins to DNA are not fully understood; however, it has been proposed that changes in double-strand conformations, such as stretching, bending, and over-twisting, as well as local variations in DNA stiffness/flexibility may play a role. The present work investigates, at the single molecule level, the morphological consequences of DNA methylation in vitro. By tracking the atomic force microscopy images of single DNA molecules, we characterize DNA conformations pertaining to two different degrees of methylation. In particular, we observe that methylation induces no relevant variations in DNA contour lengths, but produces measurable incremental changes in persistence lengths. Furthermore, we observe that for the methylated chains, the statistical distribution of angles along the DNA coordinate length is characterized by a double exponential decay, in agreement with what is predicted for polyelectrolytes. The results reported herein support the claim that the biological consequences of the methylation process, specifically difficulties in protein-DNA binding, are at least partially due to DNA conformation modifications.

Biophysical characterization of a binding site for TLQP-21, a naturally occurring peptide which induces resistance to obesity.

Recently, we demonstrated that TLQP-21 triggers lipolysis and induces resistance to obesity by reducing fat accumulation [1]. TLQP-21 is a 21 amino acid peptide cleavage product of the neuroprotein VGF and was first identified in rat brain. Although TLQP-21 biological activity and its molecular signaling is under active investigation, a receptor for TLQP-21 has not yet been characterized. We now demonstrate that TLQP-21 stimulates intracellular calcium mobilization in CHO cells. Furthermore, using Atomic Force Microscopy (AFM), we also provide evidence of TLQP-21 binding-site characteristics in CHO cells. AFM was used in force mapping mode equipped with a cantilever suitably functionalized with TLQP-21. Attraction of this functionalized probe to the cell surface was specific and consistent with the biological activity of TLQP-21; by contrast, there was no attraction of a probe functionalized with biologically inactive analogues. We detected interaction of the peptide with the binding-site by scanning the cell surface with the cantilever tip. The attractive force between TLQP-21 and its binding site was measured, statistically analyzed and quantified at approximately 40 pN on average, indicating a single class of binding sites. Furthermore we observed that the distribution of these binding sites on the surface was relatively uniform.

Thermodynamic relation between voltage-concentration dependence and salt adsorption in electrochemical cells.

Electrochemical cells containing two electrodes dipped in an ionic solution are widely used as charge accumulators, either with polarizable (supercapacitor) or nonpolarizable (battery) electrodes. Recent applications include desalination ("capacitive deionization") and energy extraction from salinity differences ("capacitive mixing"). In this Letter, we analyze a general relation between the variation of the electric potential as a function of the concentration and the salt adsorption. This relation comes from the evaluation of the electrical and mechanical energy exchange along a reversible cycle, which involves salt adsorption and release by the electrodes. The obtained relation thus describes a connection between capacitive deionization and capacitive mixing. We check this relation with experimental data already reported in the literature, and moreover by some classical physical models for electrodes, including polarizable and nonpolarizable electrodes. The generality of the relation makes it very useful in the study of the properties of the electric double layer.

Single-molecule study of the DNA denaturation phase transition in the force-torsion space.

We use the "magnetic tweezers" technique to show the structural transitions that the DNA undergoes in the force-torsion space. In particular, we focus on the regions corresponding to negative supercoiling. These regions are characterized by the formation of the so-called denaturation bubbles, which play an essential role in the replication and transcription of DNA. We experimentally map the region of the force-torsion space where the denaturation takes place. We observe that large fluctuations in DNA extension occur at one of the boundaries of this region, i.e., when the formation of denaturation bubbles and of plectonemes compete. To describe the experiments, we introduce a suitable extension of the classical model. The model correctly describes the position of the denaturation regions, the transition boundaries, and the measured values of the DNA extension fluctuations.

Photon correlation spectroscopy with incoherent light.

Photon correlation spectroscopy (PCS) is based on measuring the temporal correlation of the light intensity scattered by the investigated sample. A typical setup requires a temporally coherent light source. Here, we show that a short-coherence light source can be used as well, provided that its coherence properties are suitably modified. This results in a "skewed-coherence" light beam allowing that restores the coherence requirements. This approach overcomes the usual need for beam filtering, which would reduce the total brightness of the beam.

Cooperative effects on the compaction of DNA fragments by the nucleoid protein H-NS and the crowding agent PEG probed by Magnetic Tweezers.

BACKGROUND: DNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. METHODS: We apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. RESULTS: In the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. CONCLUSIONS: We observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. GENERAL SIGNIFICANCE: Our data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes.

Nanoparticle characterization by using tilted laser microscopy: back scattering measurement in near field.

By using scattering in near field techniques, a microscope can be easily turned into a device measuring static and dynamic light scattering, very useful for the characterization of nanoparticle dispersions. Up to now, microscopy based techniques have been limited to forward scattering, up to a maximum of 30 degrees . In this paper we present a novel optical scheme that overcomes this limitation, extending the detection range to angles larger than 90 degrees (back-scattering). Our optical scheme is based on a microscope, a wide numerical aperture objective, and a laser illumination, with the collimated beam positioned at a large angle with respect to the optical axis of the objective (Tilted Laser Microscopy, TLM). We present here an extension of the theory of near field scattering, which usually applies only to paraxial scattering, to our strongly out-of-axis situation. We tested our instrument and our calculations with calibrated spherical nanoparticles of several different diameters, performing static and dynamic scattering measurements up to 110 degrees . The measured static spectra and decay times are compatible with the Mie theory and the diffusion coefficients provided by the Stokes-Einstein equation. The ability of performing backscattering measurements with this modified microscope opens the way to new applications of scattering in near field techniques to the measurement of systems with strongly angle dependent scattering.

Characterization of anisotropic nano-particles by using depolarized dynamic light scattering in the near field.

Light scattering techniques are widely used in many fields of condensed and soft matter physics. Usually these methods are based on the study of the scattered light in the far field. Recently, a new family of near field detection schemes has been developed, mainly for the study of small angle light scattering. These techniques are based on the detection of the light intensity near to the sample, where light scattered at different directions overlaps but can be distinguished by Fourier transform analysis. Here we report for the first time data obtained with a dynamic near field scattering instrument, measuring both polarized and depolarized scattered light. Advantages of this procedure over the traditional far field detection include the immunity to stray light problems and the possibility to obtain a large number of statistical samples for many different wave vectors in a single instantaneous measurement. By using the proposed technique we have measured the translational and rotational diffusion coefficients of rod-like colloidal particles. The obtained data are in very good agreement with the data acquired with a traditional light scattering apparatus.

Field-induced clearing in sphere-sphere and rod-sphere binary mixtures of charged colloidal particles.

Binary mixtures of larger and smaller colloids having charges of equal sign display unusual response to low-frequency electric fields. We show here that the previously reported negative torque acting on rodlike particles when in presence of a sea of smaller particles is accompanied by a field-induced clearing of the suspension. Measurements of transmitted intensity performed on mixtures of large (dilute) and small (semidilute) spherical particles indicate that such a clearing effect is universal and due to an anisotropic E2 -dependent redistribution of the small spheres around the large ones. We interpret this behavior as resulting from O(E2) electro-osmotic flows whose magnitude is greatly enhanced by the presence of the small particles.

Translocation pathways for inhaled asbestos fibers.

We discuss the translocation of inhaled asbestos fibers based on pulmonary and pleuro-pulmonary interstitial fluid dynamics. Fibers can pass the alveolar barrier and reach the lung interstitium via the paracellular route down a mass water flow due to combined osmotic (active Na+ absorption) and hydraulic (interstitial pressure is subatmospheric) pressure gradient. Fibers can be dragged from the lung interstitium by pulmonary lymph flow (primary translocation) wherefrom they can reach the blood stream and subsequently distribute to the whole body (secondary translocation). Primary translocation across the visceral pleura and towards pulmonary capillaries may also occur if the asbestos-induced lung inflammation increases pulmonary interstitial pressure so as to reverse the trans-mesothelial and trans-endothelial pressure gradients. Secondary translocation to the pleural space may occur via the physiological route of pleural fluid formation across the parietal pleura; fibers accumulation in parietal pleura stomata (black spots) reflects the role of parietal lymphatics in draining pleural fluid. Asbestos fibers are found in all organs of subjects either occupationally exposed or not exposed to asbestos. Fibers concentration correlates with specific conditions of interstitial fluid dynamics, in line with the notion that in all organs microvascular filtration occurs from capillaries to the extravascular spaces. Concentration is high in the kidney (reflecting high perfusion pressure and flow) and in the liver (reflecting high microvascular permeability) while it is relatively low in the brain (due to low permeability of blood-brain barrier). Ultrafine fibers (length < 5 mum, diameter < 0.25 mum) can travel larger distances due to low steric hindrance (in mesothelioma about 90% of fibers are ultrafine). Fibers translocation is a slow process developing over decades of life: it is aided by high biopersistence, by inflammation-induced increase in permeability, by low steric hindrance and by fibers motion pattern at low Reynolds numbers; it is hindered by fibrosis that increases interstitial flow resistances.

40-Tesla pulsed-field cryomagnet for single crystal neutron diffraction.

We present the first long-duration and high duty cycle 40-T pulsed-field cryomagnet addressed to single crystal neutron diffraction experiments at temperatures down to 2 K. The magnet produces a horizontal field in a bi-conical geometry, ±15° and ±30° upstream and downstream of the sample, respectively. Using a 1.15 MJ mobile generator, magnetic field pulses of 100 ms length are generated in the magnet, with a rise time of 23 ms and a repetition rate of 6-7 pulses per hour at 40 T. The setup was validated for neutron diffraction on the CEA-CRG three-axis spectrometer IN22 at the Institut Laue Langevin.

Coil-to-globule transition of poly(N-isopropylacrylamide) doped with chiral amino acidic comonomers.

By combined light scattering and circular dichroism measurements (CD), we have investigated the coil-to-globule transition of the thermosensitive polymer poly(N-isopropylacrylamide) (pNIPAAm) copolymerized with a 1/10 fraction of valine- or leucine-derived groups randomly positioned along the chains. The comonomers provide the pNIPAAm chains with chirality, electric charge, and increased hydrophobicity. For valine-derived copolymers, the coil-globule transition is basically unmodified with respect to pNIPAAm, whereas doping with leucine-derived groups significantly lowers the transition temperature and makes the transition discontinuous. We find the CD signal of the chiral comonomers to cleanly depend on the local chain density. We interpret this behavior as an effect of the whole chain conformation on the conformations accessible to the chiral groups.

Electrokinetics in extremely bimodal suspensions.

Prompted by the results obtained by Mantegazza et al. [Nature Physics 1 (2005) 103], where the electric birefringence of suspensions of elongated particles was strikingly affected by the presence of a sea of very small (size ratio lower than 10:1) colloidal spheres, we have undertaken an investigation of other electrokinetic phenomena in suspensions containing various relative concentrations of large (Teflon or polystyrene latex) and small (nanometer-sized silica spheres) colloids. We have determined the quantities that might be greatly affected by the size distribution of the particles, mainly in the presence of ac electric fields, since the response of the suspensions will show very characteristic relaxations, dominated in principle by the size of the particles. In this work, we report on measurements of the dielectric dispersion of mixed particles as a function of the concentration, ionic strength, and field frequency. The results indicate that the response is not just a simple combination of those obtained with suspensions of the individual particles, and in fact the presence of even small amounts of the small particles affects considerably the frequency response of the suspensions.

Optical and electro-optical derivation of the pretransitional behavior of orientational and shear viscosities in the isotropic phase of liquid crystals.

The pretransitional equilibrium properties of isotropic liquids in the proximity of the isotropic-nematic phase transition are well known and successfully modeled. Much less is known about the dynamic behavior, and in particular about the pretransitional viscosity. In this work we combine two techniques [dynamic light scattering (DLS) and electric birefringence spectroscopy (EBS)] offering complementary insights into both static and dynamic pretransitional behavior of the homologous nCB family (n-alkyl cyanobiphenyl). EBS explores the single molecule flipping dynamics retarded by a paranematic potential barrier and enables extracting the associated transport coefficient, which is found to be of Arrhenius type in the whole temperature range explored. DLS reflects the collective dynamics of correlated domains and depends on the viscous damping of the orientational order. Such a viscosity displays Arrhenius behavior only sufficiently far from the transition temperature, with deviations growing as a power law of the appropriate reduced temperature with exponents around 0.1.

Memory effects in nematics with quenched disorder.

We present a combined experimental and Monte Carlo study of a nematic phase in the presence of quenched disorder. The turbidity of a nematic liquid crystal embedded in a porous polymer membrane is measured under different applied field conditions for field-cooled and zero-field-cooled samples. We find that a significant permanent alignment of the nematic can be induced by fields as low as 0.1 V/microm applied during the isotropic to nematic transition. An analogous effect and dependence on sample history is found by studying the order parameter of a sprinkled disorder Lebwohl-Lasher spin model, indicating that dilute quenched randomness is sufficient to produce memory effects in nematics. The large memory induced by field cooling appears to be written in the system during the transition as a result of the field action on freely oriented nematic nuclei. At lower temperature the nuclei consolidate into permanent nematic textures developed from the interaction with quenched disorder.

Nematics with quenched disorder: what is left when long range order Is disrupted?

It is now generally accepted that even low amounts of quenched disorder disrupt long-range order in anisotropic systems with continuous symmetry. However, very little is known on the key item of the nature of the residual order, if any, and particularly if this has quasi-long-range or truly-short-range character. Here we address this problem both experimentally for the nematic 6CB in dilute aerosils and with computer simulations. We find that the residual order is short ranged and scales with disorder density in agreement with the Imry-Ma argument.

[Use of echography as a diagnostic aid in pediatric abdominal pathology]. / L'impiego dell'ecografia quale ausilio diagnostico nella patologia addominale pediatrica.

Echotomography was used in the diagnosis of abdominal disease in children. The results obtained were compared with those given by conventional methods. The soundness of echotomography in the location and indication of the size and structure of lesions was demonstrated in a series of 20 cases.

[Use of echography as a diagnostic aid in pediatric abdominal pathology]. / L'impiego dell'ecografia quale ausilio diagnostico in patologia pediatrica addominale

Echotomography was used in the diagnosis of abdominal paediatric pathology and results compared with traditional methods of investigation. Out of 15 cases examined, echotomography proved its value in the location of the lesions and in the definition of their dimensions and structure.

Capacitive mixing for the extraction of energy from salinity differences: survey of experimental results and electrochemical models.

The "capacitive mixing" (CAPMIX) technique is an emerging technology aimed at the extraction of energy from salinity differences, e.g. between sea and river waters. CAPMIX benefits from the voltage rise that takes place between two electrodes dipped in a saline solution when its salt concentration is changed. Several kinds of electrodes have been proposed so far: activated carbon materials (Brogioli, 2009), membrane-based ion-selective electrodes (Sales et al., 2010), and battery electrodes (Biesheuvel and van der Wal, 2010). The power production mainly depends on two properties of each single electrode: the amplitude of the potential rise upon salinity change, and the potential in the high-salinity solution. The various electrode materials that have been used returned different values of the two parameters, and hence to different power productions. In this paper, we apply electrokinetic and electrochemical models to qualitatively explain the experimentally observed behaviors of various materials under different experimental conditions. The analysis allows to devise techniques for tailoring new materials, particularly suited for the CAPMIX technique.