Francesco Valle

Scaling exponents and probability distributions of DNA end-to-end distance

The scaling of the average gyration radius of polymers as a function of their length can be experimentally determined from ensemble measurements, such as light scattering, and agrees with analytical estimates. Ensemble techniques, yet, do not give access to the full probability distributions. Single molecule techniques, instead, can deliver information on both average quantities and distribution functions. Here we exploit the high resolution of atomic force microscopy over long DNA molecules adsorbed on a surface to measure the average end-to-end distance as a function of the DNA length, and its full distribution function. We find that all the scaling exponents are close to the predicted 3D values (upsilon=0.589+/-0.006 and delta=2.58+/-0.77). These results suggest that the adsorption process is akin to a geometric projection from 3D to 2D, known to preserve the scaling properties of fractal objects of dimension df<2.

Fractal dimension and localization of DNA knots

The scaling properties of DNA knots of different complexities were studied by atomic force microscope. Following two different protocols DNA knots are adsorbed onto a mica surface in regimes of (i) strong binding, that induces a kinetic trapping of the three-dimensional (3D) configuration, and of (ii) weak binding, that permits (partial) relaxation on the surface. In (i) the radius of gyration of the adsorbed DNA knot scales with the 3D Flory exponent nu approximately 0.60 within error. In (ii), we find nu approximately 0.66, a value between the 3D and 2D (nu=3/4) exponents. Evidence is also presented for the localization of knot crossings in 2D under weak adsorption conditions.

Micro- and nanopatterning by lithographically controlled wetting

This protocol describes how to perform lithographically controlled wetting (LCW). LCW enables large-area patterning of microstructures and nanostructures of soluble materials, either organic or inorganic, including biological compounds in buffer solutions or compounds for cell guidance. LCW exploits the capillary forces of menisci established under the protrusions of a stamp placed in contact with a liquid film. In the space confined by each meniscus, the self-organization of the deposited solute yields highly ordered structures that replicate the motif of the stamp protrusions. The method does not require any particular infrastructure and can be accomplished by using simple tools such as compact discs or microscopy grids. Compared with other printing methods, LCW is universal for soluble materials, as it does not require chemical binding or other specific interactions between the solute and the surface. A process cycle takes from 2 to 36 h to be completed, depending on the choice of materials.

Effect of Fe 3 O 4 magnetic nanoparticles on lysozyme amyloid aggregation

Peptide amyloid aggregation is a hallmark of several human pathologies termed amyloid diseases. We have investigated the effect of electrostatically stabilized magnetic nanoparticles of Fe(3)O(4) on the amyloid aggregation of lysozyme, as a prototypical amyloidogenic protein. Thioflavin T fluorescence assay and atomic force microscopy were used for monitoring the inhibiting and disassembly activity of magnetic nanoparticles of Fe(3)O(4). We have found that magnetic Fe(3)O(4) nanoparticles are able to interact with lysozyme amyloids in vitro leading to a reduction of the amyloid aggregates, thus promoting depolymerization; the studied nanoparticles also inhibit lysozyme amyloid aggregation. The ability to inhibit lysozyme amyloid formation and promote lysozyme amyloid disassembly exhibit concentration-dependent characteristics with IC50 = 0.65 mg ml(-1) and DC50 = 0.16 mg ml(-1) indicating that nanoparticles interfere with lysozyme aggregation already at stoichiometric concentrations. These features make Fe(3)O(4) nanoparticles of potential interest as therapeutic agents against amyloid diseases and their non-risk exploitation in nanomedicine and nanodiagnostics.

Kinetic characterization of amyloid-beta 1-42 aggregation with a multimethodological approach.

Extensive evidence suggests that the self-assembly of amyloid-beta peptide (Aβ) is a nucleation-dependent process that involves the formation of several oligomeric intermediates. Despite neuronal toxicity being recently related to Aβ soluble oligomers, results from aggregation studies are often controversial, mainly because of the low reproducibility of several experimental protocols. Here a multimethodological study that included atomic force microscopy (AFM), transmission electron microscopy (TEM), fluorescence microscopy (FLM), mass spectrometry techniques (matrix-assisted laser desorption/ionization time-of-flight [MALDI-TOF] and electrospray ionization quadrupole time-of-flight [ESI-QTOF]), and direct thioflavin T (ThT) fluorescence spectroscopy were enabled to set up a reliable and highly reproducible experimental protocol for the characterization of the morphology and dimension of Aβ 1-42 (Aβ42) aggregates along the self-assembly pathway. This multimethodological approach allowed elucidating the diverse assembly species formed during the Aβ aggregation process and was applied to the detailed investigation of the mechanism of Aβ42 inhibition by myricetin. In particular, a very striking result was the molecular weight determination of the initial oligomeric nuclei by MALDI-TOF, composed of up to 10 monomers, and their morphology by AFM.

Self-organization of functional materials in confinement.

This Account aims to describe our experience in the use of patterning techniques for addressing the self-organization processes of materials into spatially confined regions on technologically relevant surfaces. Functional properties of materials depend on their chemical structure, their assembly, and spatial distribution at the solid state; the combination of these factors determines their properties and their technological applications. In fact, by controlling the assembly processes and the spatial distribution of the resulting structures, functional materials can be guided to technological and specific applications. We considered the principal self-organizing processes, such as crystallization, dewetting and phase segregation. Usually, these phenomena produce defective molecular films, compromising their use in many technological applications. This issue can be overcome by using patterning techniques, which induce molecules to self-organize into well-defined patterned structures, by means of spatial confinement. In particular, we focus our attention on the confinement effect achieved by stamp-assisted deposition for controlling size, density, and positions of material assemblies, giving them new chemical/physical functionalities. We review the methods and principles of the stamp-assisted spatial confinement and we discuss how they can be advantageously exploited to control crystalline order/orientation, dewetting phenomena, and spontaneous phase segregation. Moreover, we highlight how physical/chemical properties of soluble functional materials can be driven in constructive ways, by integrating them into operating technological devices.

Effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability

Changes in DNA supercoiling might be essential to generate the response of cellular machinery to temperature stress. The heat‐induced structural transition for a topoisomer depends on the value of its specific linking difference. We detect only less negatively supercoiled DNA and an abundance of alternative irregular DNA forms at culture temperatures close to the growth limit of Escherichia coli. We show that the irregular forms are derived from regular plasmid DNAs and their population in the cells is temperature‐dependent. Here, we show that it is possible to isolate and characterize individual DNA topoisomers directly from cells without a topoisomerase treatment. Temperature gradient gel electrophoresis (TGGE) and atomic force microscopy (AFM) were used to study the effect of bacteria growth temperature on the distribution of supercoiled DNA and its thermal stability.

Interactive measurement and characterization of DNA molecules by analysis of AFM images

In the past few years, computer‐based analysis of atomic‐force microscopic images has acquired increasing importance for studying biomolecules such as DNA. On the one hand, fully automated methods do not allow analysis of complex shapes; on the other hand, manual methods are usually time consuming and inaccurate. The semiautomated approach presented in this report overcomes the drawbacks of both methods.

Structure-activity relationship of acridine derivatives to amyloid aggregation of lysozyme.

BACKGROUND Amyloid-related diseases (such as Alzheimers disease or diabetes type II) are associated with self-assembly of protein into amyloid aggregates. METHODS Spectroscopic and atomic force microscopy were used to determine the ability of acridines to affect amyloid aggregation of lysozyme. RESULTS We have studied the effect of acridine derivatives on the amyloid aggregation of lysozyme to investigate the acridine structure-activity relationship. The activity of the effective planar acridines was characterized by the half-maximum depolymerization concentration DC(50) and half-maximal inhibition concentration IC(50). For the most effective acridine derivatives we examined their interaction with DNA and their effect on cell viability in order to investigate their eventual influence on cells. We thus identified planar acridine derivatives with intensive anti-amyloid activity (IC(50) and DC(50) values in micromolar range), low cytotoxicity and weak ability to interfere with the processes in the cell. CONCLUSIONS Our findings indicate that both the planarity and the tautomerism of the 9-aminoacridine core together with the reactive nucleophilic thiosemicarbazide substitution play an important role in the anti-amyloid activities of studied derivatives. GENERAL SIGNIFICANCE The present findings favor the application of the selected active planar acridines in the treatment of amyloid-related diseases.

Extracellular production of tellurium nanoparticles by the photosynthetic bacterium Rhodobacter capsulatus.

The toxic oxyanion tellurite (TeO3(2-)) is acquired by cells of Rhodobacter capsulatus grown anaerobically in the light, via acetate permease ActP2 and then reduced to Te(0) in the cytoplasm as needle-like black precipitates. Interestingly, photosynthetic cultures of R. capsulatus can also generate Te(0) nanoprecipitates (TeNPs) outside the cells upon addition of the redox mediator lawsone (2-hydroxy-1,4-naphtoquinone). TeNPs generation kinetics were monitored to define the optimal conditions to produce TeNPs as a function of various carbon sources and lawsone concentration. We report that growing cultures over a 10 days period with daily additions of 1mM tellurite led to the accumulation in the growth medium of TeNPs with dimensions from 200 up to 600-700 nm in length as determined by atomic force microscopy (AFM). This result suggests that nucleation of TeNPs takes place over the entire cell growth period although the addition of new tellurium Te(0) to pre-formed TeNPs is the main strategy used by R. capsulatus to generate TeNPs outside the cells. Finally, X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FT-IR) analysis of TeNPs indicate they are coated with an organic material which keeps the particles in solution in aqueous solvents.