Infrared nanospectroscopy of individual extracellular microvesicles

Extracellular vesicles are membrane-delimited structures, involved in several inter-cellular communication processes, both physiological and pathological, since they deliver complex biological cargo. Extracellular vesicles have been identified as possible biomarkers of several pathological diseases; thus, their characterization is fundamental in order to gain a deep understanding of their function and of the related processes. Traditional approaches for the characterization of the molecular content of the vesicles require a large quantity of sample, thereby providing an average molecular profile, while their heterogeneity is typically probed by non-optical microscopies that, however, lack the chemical sensitivity to provide information of the molecular cargo. Here, we perform a study of individual microvesicles, a subclass of extracellular vesicles generated by the outward budding of the plasma membrane, released by two cultures of glial cells under different stimuli, by applying a state-of-the-art infrared nanospectroscopy technique based on the coupling of an atomic force microscope and a pulsed laser, which combines the label-free chemical sensitivity of infrared spectroscopy with the nanometric resolution of atomic force microscopy. By correlating topographic, mechanical and spectroscopic information of individual microvesicles, we identified two main populations in both families of vesicles released by the two cell cultures. Subtle differences in terms of nucleic acid content among the two families of vesicles have been found by performing a fitting procedure of the main nucleic acid vibrational peaks in the 1000–1250 cm-1 frequency range

Free Meixner states

Free Meixner states are a class of functionals on non-commutative polynomials introduced in math.CO/0410482. They are characterized by a resolvent-type form for the generating function of their orthogonal polynomials, by a recursion relation for those polynomials, or by a second-order non-commutative differential equation satisfied by their free cumulant functional. In this paper, we construct an operator model for free Meixner states. By combinatorial methods, we also derive an operator model for their free cumulant functionals. This, in turn, allows us to construct a number of examples. Many of these examples are shown to be trivial, in the sense of being free products of functionals which depend on only a single variable, or rotations of such free products. On the other hand, the multinomial distribution is a free Meixner state and is not a product. Neither is a large class of tracial free Meixner states which are analogous to the simple quadratic exponential families in statistics.Comment: 30 page

Myoblast adhesion, proliferation and differentiation on Human Elastin-Like Polypeptide (HELP) hydrogels

Purpose: The biochemical, mechanical and topographic properties of extracellular matrix are crucially involved in determining skeletal muscle cells morphogenesis, proliferation and differentiation. Human elastin-like polypeptides (HELPs) are recombinant biomimetic proteins designed to mimicking some properties of the native matrix protein; when employed as myoblasts adhesion substrates they stimulate in vitro myogenesis. Given the consequences that biophysical properties of extracellular matrix exert on skeletal muscle cells, the aim of this work was to investigate the effects of HELP hydrogels on myoblasts viability and functions. Methods: We recently synthesized a novel polypeptide, HELPc, by fusing the elastin-like backbone to a 41aa stretch present in the α2 chain of type IV collagen, containing two RGD motifs. To obtain hydrogels, the enzymatic cross-linking of the HELPc was accomplished by transglutaminase. Here, we employed both non cross-linked HELPc glass coatings and cross-linked HELPc hydrogels at different monomer density as adhesion substrates for C2C12 cells, used as myoblasts model. Results: By comparing cell adhesion, proliferation and differentiation, we revealed several striking differences. Depending on support rigidity, adhesion to HELPc substrates dictates cell morphology, spreading, focal adhesions formation and cytoskeletal organization. Hydrogels greatly stimulated cell proliferation, particularly in low serum-medium, and partially inhibited myogenic differentiation. Conclusions: In the whole, the results underline the potentiality of these genetically engineered polypeptides as a tool for dissecting crucial steps in myogenesis

Influence of steps on the tilting and adsorption dynamics of ordered Pn films on vicinal Ag(111) surfaces

Here we present a structural study of pentacene (Pn) thin films on vicinal Ag(111) surfaces by He atom diffraction measurements and density functional theory (DFT) calculations supplemented with van der Waals (vdW) interactions. Our He atom diffraction results suggest initial adsorption at the step edges evidenced by initial slow specular reflection intensity decay rate as a function of Pn deposition time. In parallel with the experimental findings, our DFT+vdW calculations predict the step edges as the most stable adsorption site on the surface. An isolated molecule adsorbs as tilted on the step edge with a binding energy of 1.4 eV. In addition, a complete monolayer (ML) with pentacenes flat on the terraces and tilted only at the step edges is found to be more stable than one with all lying flat or tilted molecules, which in turn influences multilayers. Hence our results suggest that step edges can trap Pn molecules and act as nucleation sites for the growth of ordered thin films with a crystal structure similar to that of bulk Pn.Comment: 4 pages, 4 figures, 1 tabl

Electro-chemical deposition of zinc oxide nanostructures by using two electrodes

One of the most viable ways to grow nanostructures is electro deposition. However, most electrodeposited samples are obtained by three-electrode electrochemical cell. We successfully use a much simpler two-electrode cell to grow different ZnO nanostructures from common chemical reagents. Concentration, pH of the electrolytes and growth parameters like potentials at the electrodes, are tailored to allow fast growth without complexity. Morphology and surface roughness are investigated by Scanning Electron and Air Force Microscopy (SEM and AFM) respectively, crystal structure by X-Ray Diffraction measurements (XRD) and ZnO stoichiometry by core level photoemission spectroscopy (XPS)

Computational Evolution of Beta-2-Microglobulin Binding Peptides for Nanopatterned Surface Sensors

The bottom-up design of smart nanodevices largely depends on the accuracy by which each of the inherent nanometric components can be functionally designed with predictive methods. Here, we present a rationally designed, self-assembled nanochip capable of capturing a target protein by means of pre-selected binding sites. The sensing elements comprise computationally evolved peptides, designed to target an arbitrarily selected binding site on the surface of beta-2-Microglobulin (β2m), a globular protein that lacks well-defined pockets. The nanopatterned surface was generated by an atomic force microscopy (AFM)-based, tip force-driven nanolithography technique termed nanografting to construct laterally confined self-assembled nanopatches of single stranded (ss)DNA. These were subsequently associated with an ssDNA–peptide conjugate by means of DNA-directed immobilization, therefore allowing control of the peptide’s spatial orientation. We characterized the sensitivity of such peptide-containing systems against β2m in solution by means of AFM-based differential topographic imaging and surface plasmon resonance (SPR) spectroscopy. Our results show that the confined peptides are capable of specifically capturing β2m from the surface–liquid interface with micromolar affinity, hence providing a viable proof-of-concept for our approach to peptide design

Glioma-associated stem cells: A novel class of tumor-supporting cells able to predict prognosis of human low-grade gliomas.

Background: Translational medicine aims at transferring advances in basic science research into new approaches for diagnosis and treatment of diseases. Low-grade gliomas (LGG) have a heterogeneous clinical behavior that can be only partially predicted employing current state-of-the-art markers, hindering the decision-making process. To deepen our comprehension on tumor heterogeneity, we dissected the mechanism of interaction between tumor cells and relevant components of the neoplastic environment, isolating, from LGG and high-grade gliomas (HGG), proliferating stem cell lines from both the glioma stroma and, where possible, the neoplasm. Methods and Findings: We isolated glioma-associated stem cells (GASC) from LGG (n=40) and HGG (n=73). GASC showed stem cell features, anchorage-independent growth, and supported the malignant properties of both A172 cells and human glioma-stem cells, mainly through the release of exosomes. Finally, starting from GASC obtained from HGG (n=13) and LGG (n=12) we defined a score, based on the expression of 9 GASC surface markers, whose prognostic value was assayed on 40 subsequent LGG-patients. At the multivariate Cox analysis, the GASC-based score was the only independent predictor of overall survival and malignant progression free-survival. Conclusions: The microenvironment of both LGG and HGG hosts non-tumorigenic multipotent stem cells that can increase in vitro the biological aggressiveness of glioma-initiating cells through the release of exosomes. The clinical importance of this finding is supported by the strong prognostic value associated with the characteristics of GASC. This patient-based approach can provide a groundbreaking method to predict prognosis and to exploit novel strategies that target the tumor stroma