M. De Spirito

Small- and wide-angle elastic light scattering study of fibrin structure

We show how small- and wide-angle elastic light scattering (q ∼ 0.03-30 μm -1 ) can be used to quantitatively characterize the structure of polymeric gels made of semi-flexible entangled fibers. We applied the technique to the study of fibrin gels grown from the polymerization of fibrinogen (FG) macromolecular monomers following activation by the enzyme thrombin (TH), at different concentrations and under different physical-chemical conditions of the gelling solution. Our findings show that the gel can be imagined as a random network of fibers of size d and density p, entangled together to form densely packed blobs of mass fractal dimension D m and average size ξ, which may overlap by a factor η and exhibit a long-range order. Provided that d ≥50-100 nm, all of the above parameters can be recovered by the use of a global fitting function developed by us on the basis on the proposed gel model. When the fibers are thinner (d < ∼50 nm), only the fiber mass/length ratio μ ∼ ρd 2 can be retrieved instead of d and p. Our data confirm and quantify the major changes in the gel structure that can be obtained by varying either the salt concentration of the solution and/or the molar ratio TH/FG. Gels formed in Tris-HCl 50 mM/NaCl 150 mM, pH 7.4 and TH/FG = 0.01 are characterized by relatively small, fairly branched (D in ∼ 1.4-2.0) fibers with a mass/length ratio independent of concentration. On reducing the TH/FG ratio, the fibers become increasingly thicker, with d ∼ 90 nm at TH/FG = 10 -5 . When the salt concentration is reduced to NaCl 100 mM (TH/FG = 0.01) the fibers are less branched (D m ∼ 1.2-1.4), but much thicker, with μ increasing by an order of magnitude. These effects are quantitatively analyzed and discussed.

Fluctuations and the Rate-Limiting Step of Peptide-Induced Membrane Leakage

Peptide-induced vesicle leakage is a common experimental test for the membrane-perturbing activity of antimicrobial peptides. The leakage kinetics is usually very slow, requiring minutes to hours for complete release of vesicle contents, and exhibits a biphasic behavior. We report here that, in the case of the peptaibol trichogin GA IV, all processes involved in peptide-membrane interaction, such as peptide-membrane association, peptide aggregation, and peptide translocation, take place on a timescale much shorter than the leakage kinetics. On the basis of these findings, we propose a stochastic model in which the leakage kinetics is determined by the discrete nature of a vesicle suspension: peptides are continuously exchanging among vesicles, producing significant fluctuations over time in the number of peptide molecules bound to each vesicle, and in the formation of pores. According to this model, the fast initial leakage is caused by vesicles that contain at least one pore after the peptides are randomly distributed among the liposomes, whereas the slower release is associated with the time needed to occasionally reach in an intact vesicle the critical number of bound peptides necessary for pore formation. Fluctuations due to peptide exchange among vesicles therefore represent the rate-limiting step of such a slow mechanism.

Self-similarity properties of alpha-crystallin supramolecular aggregates

The supramolecular aggregation of alpha-crystallin, the major protein of the eye lens, was investigated by means of static and dynamic light scattering. The aggregation was induced by generating heat-modified alpha-crystallin forms and by stabilizing the clusters with calcium ions. The kinetic pattern of the aggregation and the structural features of the clusters can be described according to the reaction limited cluster-cluster aggregation theory previously adopted for the study of colloidal particles aggregation systems. Accordingly, the average mass and the hydrodynamic radius of alpha-crystallin supramolecular aggregates grow exponentially in time. The structure factor of the clusters is typical of fractal aggregates. A fractal dimension df approximately 2.15 was determined, indicating a low probability of sticking together of the primitive aggregating particles. As a consequence, the slow-forming clusters assemble a rather compact structure. The basic units forming the fractal aggregates were found to have a radius about twice (approximately 17 nm) that of the native protein and 5.3 times its size, which is consistent with an intermediate molecular assembly corresponding to the already known high molecular weight forms of alpha-crystallin.

Intervillous circulation in intra-uterine growth restriction. Correlation to fetal well being

Magnetic resonance imaging requested for a potentially serious indication, provided a unique opportunity to explore the intervillous circulation of placentas from pregnancies complicated by Intra Uterine Growth Restriction (IUGR) and to compare them to normal cases. This allowed an innovative characterization of in vivo utero-placental blood flow, correlating a compromised intervillous circulation in IUGR to the deterioration of fetal condition. MR imaging was requested to rule out suspected posterior placental adhesive disorders in 26 patients. Twelve patients had fetuses appropriate for gestational age, while in 14 patients fetuses were affected by severe IUGR. Multiphasic dynamic contrast-enhanced sagittal sequences were acquired and a quantitative analysis of signal intensity and enhancement kinetics was performed for both the entire placenta and for selected regions. Images disclosed a homogeneous perfusion overall the placenta in normal cases, while IUGR placentas displayed a slow intervillous blood flow, along with many patchy unperfused areas. Intermittent stops worsen the perfusion dynamics of the intervillous mostly in IUGR cases with an elevated ductus venosus pulsatility index. In conclusion, we proved that in IUGR placenta maternal placental blood flow is extremely compromised and that superimposed dynamic phenomena concur to worsen the intervillous circulation leading to an end-stage fetal decompensation.

Investigation of the spatial distribution of glutathione redox-balance in live cells by using Fluorescence Ratio Imaging Microscopy

The dynamics of redox elements in biologic systems is a major challenge for redox signaling and oxidative stress research. Oxidative stress or signaling events can affect sulfur switches differently, thus creating a variation in the spatial distribution of these redox states, which therefore act simultaneously as regulators and indicators of key cellular functions in both physiological and pathological settings. A gluthatione specific redox-sensitive protein (i.e. a mutant of the Yellow Fluorescent Protein (rxYFP)) has been found to equilibrate in vivo with the gluthatione/gluthatione disulfide (GSH:GSSG) redox couple. rxYFP, employed ratiometrically, allows to generate high resolution maps of the fraction of the reduced protein (R) inside a cell. Here we developed an analytical procedure able to investigate intracellular changes in the glutathione redox-balance, which can occur in live mammalian cells, based on the deconvolution of the histogram of redox maps of 293-TPhoenix human embryonic kidney cells. The intracellular spatial distributions of oxidized and reduced elements have been discriminated. Finally, by transfecting cells with human Glutaredoxin V (GRX-V), an enzyme deputed to maintain reduced the thiol groups of their partner proteins, we can disclose that the significant shift towards more reduced state, with respect to that recovered from non-transfected cells, consists, instead, in a shift towards reduced values of the high R region (reduced), while leaving unaltered the glutathione redox-balance of the intracellular side of the plasma membrane.

Recent advances in superhydrophobic surfaces and their relevance to biology and medicine.

By mimicking naturally occurring superhydrophobic surfaces, scientists can now realize artificial surfaces on which droplets of a few microliters of water are forced to assume an almost spherical shape and an extremely high contact angle. In recent decades, these surfaces have attracted much attention due to their technological applications for anti-wetting and self-cleaning materials. Very recently, researchers have shifted their interest to investigate whether superhydrophobic surfaces can be exploited to study biological systems. This research effort has stimulated the design and realization of new devices that allow us to actively organize, visualize and manipulate matter at both the microscale and nanoscale levels. Such precise control opens up wide applications in biomedicine, as it allows us to directly manipulate objects at the typical length scale of cells and macromolecules. This progress report focuses on recent biological and medical applications of superhydrophobicity. Particular regard is paid to those applications that involve the detection, manipulation and study of extremely small quantities of molecules, and to those that allow high throughput cell and biomaterial screening.

Self-assembling of large ordered DNA arrays using superhydrophobic patterned surfaces

In this paper we present a simple and robust method to realize highly ordered arrays of stretched and suspended DNA molecules over the millimeter length scale. To this end we used an ad hoc designed superhydrophobic surface made of high aspect-ratio silicon pillars, where we deposited a droplet containing genomic DNA. A precise positioning of DNA strands was achieved by shaping the silicon pillars so that sharpened features resembling tips were included. Such features allowed us to accurately control the droplet de-wetting dynamics, pinning DNA strands in a well-defined position above pillars. The proposed technique has the potential to positively impact on the development of novel DNA chips for genetic analysis.

A hybrid characterization framework to determine the visco-hyperelastic properties of a porcine zona pellucida

The zona pellucida (ZP) is a specialized extracellular matrix surrounding the developing oocyte. This thick matrix consists of various types of glycoprotein that play different roles in the fertilization process. Nowadays, several techniques are available for assessing ZPs mechanical response. The basic assumption behind these methods is that the ZP behaves like an elastic body: hence, dissipative forces are neglected and Youngs modulus remains unaffected by probe dynamics. However, dissipative forces are strongly regulated by the slippage of ZP chains past one another while reaction forces related to elastic deformations (driven by the ability of each chain to stretch) depend on the ZP structure (i.e. number of cross-links and distances between knots). Although viscous reaction forces generated by the ZP are one of the main factors regulating sperm transit, their peculiar behaviour along the ZP structure remains poorly understood and rarely investigated. In order to overcome this limitation, a novel visco-hyperelastic model describing the porcine ZP reaction forces generated by nanoindentations at different probe rates is developed and verified in this study. Visco-hyperelastic parameters of porcine ZP membranes are determined by means of a hybrid characterization framework combining atomic force microscopy nanoindentation measurements, nonlinear finite-element analysis and nonlinear optimization. Remarkably, it is possible to separate the contributions of hyperelastic and viscous terms to ZP mechanical response and evaluate the error made in the determination of ZP mechanical properties if viscous effects were not considered.

Detection of Biofilm-Grown Aspergillus fumigatus by Means of Atomic Force Spectroscopy: Ultrastructural Effects of Alginate Lyase

Aspergillus fumigatus has become a leading cause of fungal morbidity and mortality, especially in immunocompromised patients. This fungus is able to grow as a multicellular community and produce a hydrophobic extracellular matrix (ECM), mainly composed of galactomannan and α-1,3 glucans, to protect itself from host defenses and antimicrobial drugs. This matrix envelops the fungus hyphae, binding them into a contiguous sheath on the colony surface, forming a biofilm and increasing the fungal resistance to adverse environmental factors. Adherence to host cells and resistance to physical removal play a key role in fungal colonization and invasion of the host and in a wide range of infections. Here we show that, by using atomic force spectroscopy, it is possible to exploit the peculiar hydrophobicity of the biofilm components (i.e., cell walls, ECM) to detect the biofilm spread, its growth, and lysis on rough surfaces. By means of this approach, we demonstrate that alginate lyase, an enzyme known to reduce negatively charged alginate levels in microbial biofilms, reduces the biofilm adhesion forces suggesting a loss of ECM from the biofilm, which could be used to enhance pharmacological treatments.

The type 2B p.R1306W natural mutation of von Willebrand factor dramatically enhances the multimer sensitivity to shear stress.

Shear stress triggers conformational stretching of von Willebrand factor (VWF), which is responsible for its self‐association and binding to the platelet receptor glycoprotein (GP)Ibα. This phenomenon supports primary hemostasis under flow. Type 2B VWF natural mutants are considered to have increased affinity for platelet GPIbα.