Loredana Casalis

Efficient water oxidation at carbon nanotube–polyoxometalate electrocatalytic interfaces

Water is the renewable, bulk chemical that nature uses to enable carbohydrate production from carbon dioxide. The dream goal of energy research is to transpose this incredibly efficient process and make an artificial device whereby the catalytic splitting of water is finalized to give a continuous production of oxygen and hydrogen. Success in this task would guarantee the generation of hydrogen as a carbon-free fuel to satisfy our energy demands at no environmental cost. Here we show that very efficient and stable nanostructured, oxygen-evolving anodes are obtained by the assembly of an oxygen-evolving polyoxometalate cluster (a totally inorganic ruthenium catalyst) with a conducting bed of multiwalled carbon nanotubes. Our bioinspired electrode addresses the one major challenge of artificial photosynthesis, namely efficient water oxidation, which brings us closer to being able to power the planet with carbon-free fuels.

X-ray Diffraction and Computation Yield the Structure of Alkanethiols on Gold(111)

The structure of self-assembled monolayers (SAMs) of long-chain alkyl sulfides on gold(111) has been resolved by density functional theory–based molecular dynamics simulations and grazing incidence x-ray diffraction for hexanethiol and methylthiol. The analysis of molecular dynamics trajectories and the relative energies of possible SAM structures suggest a competition between SAM ordering, driven by the lateral van der Waals interaction between alkyl chains, and disordering of interfacial Au atoms, driven by the sulfur-gold interaction. We found that the sulfur atoms of the molecules bind at two distinct surface sites, and that the first gold surface layer contains gold atom vacancies (which are partially redistributed over different sites) as well as gold adatoms that are laterally bound to two sulfur atoms.

Synthesis and Characterization of a Carbon Nanotube−Dendron Series for Efficient siRNA Delivery

A new series of dendron-functionalized multiwalled carbon nanotube (MWNT) derivatives, characterized by the presence of numerous positively charged tetraalkyl ammonium salts at the periphery of the dendron, has been synthesized. The positive charges on the MWNT surface, coupled with the unique ability of carbon nanotubes (CNTs) to penetrate cell membranes, make the new derivatives potentially ideal vectors for siRNA delivery. Using a fluorescently labeled, noncoding siRNA sequence, we demonstrate that cytoplasmic delivery of the nucleic acid is remarkably increased throughout the different dendron generations. The work reported here highlights the fact that dendron-functionalized CNTs can be rationally designed as efficient carriers of siRNA that can eventually lead to gene silencing.

Toward multiprotein nanoarrays using nanografting and DNA directed immobilization of proteins

Atomic force microscopy nanografting was utilized to prepare DNA nanopatches of different sizes (200 x 200 to 1000 x 1000 nm(2)) onto which DNA-protein conjugates can be anchored through DNA-directed immobilization. Height measurements were used to assess the binding of the proteins as well as their subsequent interaction with other components, such as antibodies. The results indicate that nanografted patch arrays are well suited for application in biosensing and could enable the fabrication of multifeature protein nanoarrays.

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. Stem Cells 2014;32:1239–1253

Graphene Oxide Nanosheets Reshape Synaptic Function in Cultured Brain Networks.

Graphene offers promising advantages for biomedical applications. However, adoption of graphene technology in biomedicine also poses important challenges in terms of understanding cell responses, cellular uptake, or the intracellular fate of soluble graphene derivatives. In the biological microenvironment, graphene nanosheets might interact with exposed cellular and subcellular structures, resulting in unexpected regulation of sophisticated biological signaling. More broadly, biomedical devices based on the design of these 2D planar nanostructures for interventions in the central nervous system require an accurate understanding of their interactions with the neuronal milieu. Here, we describe the ability of graphene oxide nanosheets to down-regulate neuronal signaling without affecting cell viability.

Defined α-synuclein prion-like molecular assemblies spreading in cell culture

Backgroundα-Synuclein (α-syn) plays a central role in the pathogenesis of synucleinopathies, a group of neurodegenerative disorders that includes Parkinson disease, dementia with Lewy bodies and multiple system atrophy. Several findings from cell culture and mouse experiments suggest intercellular α-syn transfer.ResultsThrough a methodology used to obtain synthetic mammalian prions, we tested whether recombinant human α-syn amyloids can promote prion-like accumulation in neuronal cell lines in vitro. A single exposure to amyloid fibrils of human α-syn was sufficient to induce aggregation of endogenous α-syn in human neuroblastoma SH-SY5Y cells. Remarkably, endogenous wild-type α-syn was sufficient for the formation of these aggregates, and overexpression of the protein was not required.ConclusionsOur results provide compelling evidence that endogenous α-syn can accumulate in cell culture after a single exposure to exogenous α-syn short amyloid fibrils. Importantly, using α-syn short amyloid fibrils as seed, endogenous α-syn aggregates and accumulates over several passages in cell culture, providing an excellent tool for potential therapeutic screening of pathogenic α-syn aggregates.

Control of steric hindrance on restriction enzyme reactions with surface-bound DNA nanostructures.

To understand better enzyme/DNA interactions and to design innovative detectors based on DNA nanoarrays, we need to study the effect of nanometric confinement on the biochemical activity of the DNA molecules. We focus on the study of the restriction enzyme reactions (DpnII) within DNA nanostructures on flat gold films by atomic force microscopy (AFM). Typically we work with a few patches of DNA self assembled monolayers (SAMs) that are hundred nm in size and are lithographically fabricated within alkylthiol SAMs by AFM nanografting. We start by nanografting a few patches of a single-stranded DNA (ssDNA) molecule of 44 base pairs (bps) with a 4 bps recognition sequence (specific for DpnII) in the middle. Afterwards, reaction-ready DNA nanopatches are obtained by hybridization with a complementary 44bps ssDNA sequence. The enzymatic reactions were carried out over nanopatches with different density. By carrying out AFM height measurements, we are able to show that the capability of the DpnII enzyme to reach and react at the recognition site is easily varied by controlling the DNA packing in the nanostructures. We have found strong evidence that inside our ordered DNA nanostructures the enzyme (that works as a dimer) can operate down to the limit in which the space between adjacent DNA molecules is equal to the size of the DNA/enzyme complex. Similar experiments were carried out with a DNA sequence without the recognition site, clearly finding that in that case the enzymatic reaction did not lead to digestion of the molecules. These findings suggest that it is possible to tune the efficiency of an enzymatic reaction on a surface by controlling the steric hindrance inside the DNA nanopatches without vary any further physical or chemical variable. These findings are opening the door to novel applications in both the fields of biosensing and fundamental biophysics.

Two-dimensional enzyme diffusion in laterally confined DNA monolayers

Addressing the effects of confinement and crowding on biomolecular function may provide insight into molecular mechanisms within living organisms, and may promote the development of novel biotechnology tools. Here, using molecular manipulation methods, we investigate restriction enzyme reactions with double-stranded (ds)DNA oligomers confined in relatively large (and flat) brushy matrices of monolayer patches of controlled, variable density. We show that enzymes from the contacting solution cannot access the dsDNAs from the top-matrix interface, and instead enter at the matrix sides to diffuse two-dimensionally in the gap between top- and bottom-matrix interfaces. This is achieved by limiting lateral access with a barrier made of high-density molecules that arrest enzyme diffusion. We put forward, as a possible explanation, a simple and general model that relates these data to the steric hindrance in the matrix, and we briefly discuss the implications and applications of this strikingly new phenomenon.

Oriented Immobilization of Prion Protein Demonstrated via Precise Interfacial Nanostructure Measurements

Nanopatterning of biomolecules on functionalized surfaces offers an excellent route for ultrasensitive protein immobilization, for interaction measurements, and for the fabrication of devices such as protein nanoarrays. An improved understanding of the physics and chemistry underlying the device properties and the recognition process is necessary for performance optimization. This is especially important for the recognition and immobilization of intrinsically disordered proteins (IDPs), like the prion protein (PrP), a partial IDP, whose folding and stability may be influenced by local environment and confinement. Atomic force microscopy allows for both highly controllable nanolithography and for sensitive and accurate direct detection, via precise topographic measurements on ultraflat surfaces, of protein interactions in a liquid environment, thus different environmental parameters affecting the biorecognition phenomenon can be investigated in situ. Using nanografting, a tip-induced lithographic technique, and an affinity immobilization strategy based on two different histidine tagged antibodies, with high nM affinity for two different regions of PrP, we successfully demonstrated the immobilization of recombinant mouse PrP onto nanostructured surfaces, in two different orientations. Clear discrimination of the two molecular orientations was shown by differential height (i.e., topographic) measurements, allowing for the estimation of binding parameters and the full characterization of the nanoscale biorecognition process. Our work opens the way to several high sensitivity diagnostic applications and, by controlling PrP orientation, allows for the investigation of unconventional interactions with partially folded proteins, and may serve as a platform for protein misfolding and refolding studies on PrP and other thermodynamically unstable, fibril forming, proteins.