Structural analysis of intrinsically disordered proteins: computer atomistic simulation

Intrinsically disordered proteins (IDPs) are biomolecules that do not have a definite 3D structure; their role in the biochemical network of a cell relates to their ability to switch rapidly among different secondary and tertiary structures. For this reason, applying a simulation computer program to their structural study turns out to be problematic, as their dynamical simulation cannot start from a known list of atomistic positions, as is the case for globular proteins that do crystallize and that one can analyse by X-ray spectroscopy to determine their structure. We have established a method to perform a computer simulation of these proteins, apt to gather statistically significant data on their transient structures. The only required input to start the procedure is the primary sequence of the disordered domains of the protein, and the 3D structure of the ordered domains, if any. For a fully disordered protein the method is as follows: (a) The first step is the creation of a multi-rod-like configuration of the molecule, derived from its primary sequence. This structure evolves dynamically in vacuo or in an implicit model of solvent, until its gyration radius - or any other measure of the overall configuration of the molecule - reaches the experimental average value; at this point, one may follow two different paths. (b1) If the study focuses on transient secondary structures of the molecule, one puts the structure obtained at the end of the first step in a box containing solvent molecules in explicit implementation, and a standard molecular dynamics simulation follows. (b2) If the study focuses on the tertiary structure of the molecule, a larger sampling of the phase space is required, with the molecule moving in very large and diverse regions of the phase space. To this end, the structure of the IDP is let evolve dynamically in an implicit solvent using metadynamics, an algorithm that keeps track of the regions of the phase space already sampled, and forces the system to wander in further regions of the phase space. (c) One can increase the accuracy of the statistical information gathered in both cases by fitting, where available, experimental data of the protein. In this step one extracts an ensemble of ’best’ conformers from the pool of all configurations produced in the simulated dynamics. One derives this ensemble by means of an ensemble optimization method, implementing a genetic algorithm. We have applied this procedure to the simulation of tau, one of the largest fully disordered proteins, which is involved in the development of Alzheimer’s disease and of other neurodegenerative diseases. We have combined the results of our simulation with small-angle X-ray scattering experimental data to extract from the dynamics an optimized ensemble of most probable conformers of tau. The method can be easily adapted to IDPs entailing ordered domains

The Microstrain-Doping Phase Diagram of the Iron Pnictides Heterostructures at Atomic Limit

The 3D phase diagram of iron pnictides where the critical temperature depends on charge density and microstrain in the active FeAs layers is proposed. The iron pnictides superconductors are shown to be a practical realization of a heterostructure at the atomic limit made of a superlattice of FeAs layers intercalated by spacer layers. We have focussed our interest on the A 1-x BxFe2As2 (122) families and we show that FeAs layers have a tensile microstrain due to the misfit strain between the active layers and the spacers. We have identified the critical range of doping and microstrain where the critical temperature gets amplified to its maximum value.Comment: 5 pages, 3 figure

Red blood cells membrane micropolarity as a novel diagnostic indicator of type 1 and type 2 diabetes

Classification of the category of diabetes is extremely important for clinicians to diagnose and select the correct treatment plan. Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins, as well as impairment in cholesterol homeostasis, can alter lipid density, packing, and interactions of Red blood cells (RBC) plasma membranes in type 1 and type 2 diabetes, thus varying their membrane micropolarity. This can be estimated, at a submicrometric scale, by determining the membrane relative permittivity, which is the factor by which the electric field between the charges is decreased relative to vacuum. Here, we employed a membrane micropolarity sensitive probe to monitor variations in red blood cells of healthy subjects (n=16) and patients affected by type 1 (T1DM, n=10) and type 2 diabetes mellitus (T2DM, n=24) to provide a cost-effective and supplementary indicator for diabetes classification. We find a less polar membrane microenvironment in T2DM patients, and a more polar membrane microenvironment in T1DM patients compared to control healthy patients. The differences in micropolarity are statistically significant among the three groups (p<0.01). The role of serum cholesterol pool in determining these differences was investigated, and other factors potentially altering the response of the probe were considered in view of developing a clinical assay based on RBC membrane micropolarity. These preliminary data pave the way for the development of an innovative assay which could become a tool for diagnosis and progression monitoring of type 1 and type 2 diabetes. Keywords: Diabetes mellitus, Membrane micropolarity, Red blood cells, Fluorescence lifetime microscopy, Metabolic imaging, Personalized medicin

Biosynthesis and physico-chemical characterization of high performing peptide hydrogels@graphene oxide composites

: Hydrogels based on short peptide molecules are interesting biomaterials with wide present and prospective use in biotechnologies. A well-known possible drawback of these materials can be their limited mechanical performance. In order to overcome this problem, we prepared Fmoc-Phe3self-assembling peptides by a biocatalytic approach, and we reinforced the hydrogel with graphene oxide nanosheets. The formulation here proposed confers to the hydrogel additional physicochemical properties without hampering peptide self-assembly. We investigated in depth the effect of nanocarbon morphology on hydrogel properties (i.e. morphology, viscoelastic properties, stiffness, resistance to an applied stress). In view of further developments towards possible clinical applications, we have preliminarily tested the biocompatibility of the composites. Our results showed that the innovative hydrogel composite formulation based on FmocPhe3 and GO is a biomaterial with improved mechanical properties that appears suitable for the development of biotechnological applications

Plasma protein corona reduces the haemolytic activity of graphene oxide nano and micro flakes

Intensive research on the bio-applications of graphene and its derivatives is leading to many technological applications. Graphene Oxide (GO), for its unique 2-D structure and its physical/chemical properties, has attracted increasing interest over the last few years in the fields of drug/gene delivery, biological imaging and antibacterial treatments. Together with these great potentials for biomedical applications, several aspects of graphene toxicity mechanisms including oxidative stress, cutting off intracellular metabolic routes and cell membrane rupture must be carefully evaluated. In this work we demonstrate that the GO flakes, able to disrupt the erythrocyte plasma membrane, greatly reduce their hemolytic activity after interacting with plasma proteins

Gamma-ray-induced structural transformation of GQDs towards the improvement of their optical properties, monitoring of selected toxic compounds, and photo-induced effect on bacterial strains

Structural modification of different carbon-based nanomaterials is often necessary to improve their morphology and optical properties, particularly the incorporation of N-atoms in graphene quantum dots (GQDs). Here, a clean, simple, one-step, and eco-friendly method for N-doping of GQDs using gamma irradiation is reported. GQDs were irradiated in the presence of the different ethylenediamine (EDA) amounts (1 g, 5 g, and 10 g) and the highest % of N was detected in the presence of 10 g. N-doped GQDs emitted strong, blue photoluminescence (PL). Photoluminescence quantum yield was increased from 1.45, as obtained for non-irradiated dots, to 7.24% for those irradiated in the presence of 1 g of EDA. Modified GQDs were investigated as a PL probe for the detection of insecticide Carbofuran (2,2-Dimethyl-2,3-dihydro-1-benzofuran-7- yl methylcarbamate) and herbicide Amitrole (3-amino-1,2,4-triazole). The limit of detection was 5.4 µmol L−1 for Carbofuran. For the first time, Amitrole was detected by GQDs in a turn-off/turn-on mechanism using Pd(II) ions as a quenching agent. First, Pd(II) ions were quenched (turn-off) PL of GQDs, while after Amitrole addition, PL was recovered linearly with Amitrole concentration (turn-on). LOD was 2.03 µmol L−1 . These results suggest that modified GQDs can be used as an efficient new material for Carbofuran and Amitrole detection. Furthermore, the phototoxicity of dots was investigated on both Gram-positive and Gram-negative bacterial strains. When bacterial cells were exposed to different GQD concentrations and illuminated with light of 470 nm wavelength, the toxic effects were not observed

Recent advances in the label-free characterization of exosomes for cancer liquid biopsy: From scattering and spectroscopy to nanoindentation and nanodevices

Exosomes (EXOs) are nano-sized vesicles secreted by most cell types. They are abundant in bio-fluids and harbor specific molecular constituents from their parental cells. Due to these characteristics, EXOs have a great potential in cancer diagnostics for liquid biopsy and personalized medicine. Despite this unique potential, EXOs are not yet widely applied in clinical settings, with two main factors hindering their translational process in diagnostics. Firstly, conventional extraction methods are time-consuming, require large sample volumes and expensive equipment, and often do not provide high-purity samples. Secondly, characterization methods have some limitations, because they are often qualitative, need extensive labeling or complex sampling procedures that can induce artifacts. In this context, novel label-free approaches are rapidly emerging, and are holding potential to revolutionize EXO diagnostics. These methods include the use of nanodevices for EXO purification, and vibrational spectroscopies, scattering, and nanoindentation for characterization. In this progress report, we summarize recent key advances in label-free techniques for EXO purification and characterization. We point out that these methods contribute to reducing costs and processing times, provide complementary information compared to the conventional characterization techniques, and enhance flexibility, thus favoring the discovery of novel and unexplored EXO-based biomarkers. In this process, the impact of nanotechnology is systematically highlighted, showing how the effectiveness of these techniques can be enhanced using nanomaterials, such as plasmonic nanoparticles and nanostructured surfaces, which enable the exploitation of advanced physical phenomena occurring at the nanoscale level

Facile synthesis of L-cysteine functionalized graphene quantum dots as a bioimaging and photosensitive agent

Nowadays, a larger number of aggressive and corrosive chemical reagents as well as toxic solvents are used to achieve structural modification and cleaning of the final products. These lead to the production of residual, waste chemicals, which are often reactive, cancerogenic, and toxic to the environment. This study shows a new approach to the modification of graphene quantum dots (GQDs) using gamma irradiation where the usage of reagents was avoided. We achieved the incorporation of S and N atoms in the GQD structure by selecting an aqueous solution of L-cysteine as an irradiation medium. GQDs were exposed to gamma-irradiation at doses of 25, 50 and 200 kGy. After irradiation, the optical, structural, and morphological properties, as well as the possibility of their use as an agent in bioimaging and photodynamic therapy, were studied. We measured an enhanced quantum yield of photoluminescence with the highest dose of 25 kGy (21.60%). Both S- and N-functional groups were detected in all gamma-irradiated GQDs: amino, amide, thiol, and thione. Spin trap electron paramagnetic resonance showed that GQDs irradiated with 25 kGy can generate singlet oxygen upon illumination. Bioimaging on HeLa cells showed the best visibility for cells treated with GQDs irradiated with 25 kGy, while cytotoxicity was not detected after treatment of HeLa cells with gamma-irradiated GQDs

Buche quantiche di Si/SiGe : crescita e caratterizzazione

I più promettenti dispositivi per la generazione di radiazione THz sono attualmente i Laser a Cascata Quantica (QCL). Questi dispositivi, realizzati per mezzo dei semiconduttori III-V, sono laser unipolari basati sulle transizioni intersottobanda tra Quantum Well accoppiate. Recentemente ha suscitato un notevole interesse la possibilità di realizzare un laser analogo basato su silicio. Tale dispositivo presenterebbe numerosi vantaggi, specialmente da un punto di vista della produzione su larga scala, vista la completa compatibilità con la preesistente tecnologia del silicio. In questo contesto si inserisce il presente lavoro di tesi in cui viene trattato lo studio di eterostrutture di semiconduttore in Si/SiGe. In particolare sono state realizzate e caratterizzare Multi Quantum Well (MQW) con il pozzo in silicio e drogaggio di tipo n, allo scopo di investigare le transizioni ottiche tra le sottobande (ISBT) create in banda di conduzione dal confinamento quantistico degli elettroni. Le MQW sono state cresciute mediante Deposizione Chimica da Fase Vapore (CVD), l'unica tecnica compatibile con la produzione industriale, a partire da diversi tipi di substrati: un substrato virtuale (VS) e due substrati, il Silicon Germanium On Insulator (SGOI) e lo Strained Silicon On Insulator (SSOI), che rappresentano una recente innovazione nel campo dell'eteroepitassia. In particolare, questi ultimi sono caratterizzati, rispettivamente, dalla presenza di un film superficiale sottile di Si0.8Ge0.2 cubico e Si tensile, realizzato su uno strato di SiO2 isolante. Gli studi da noi effettuati su questi nuovi substrati hanno evidenziato la presenza di un fenomeno di instabilità morfologica in seguito ad annealing termici, detto agglomerazione, che induce la rottura del film superficiale planare e la formazione di isole su ossido. E' stata implementata una metodologia innovativa per determinare la temperatura critica Tc, oltre la quale si ha l'inizio del fenomeno descritto. Tale metodologia, basata su misure di XPS in-situ, presenta evidenti vantaggi rispetto a quelle tipicamente utilizzate in letteratura, basate su misure AFM, non ultima la possibilità di campionare, con un'elevata sensibilità, ampie regioni dei substrati. E' stato inoltre dimostrato che la presenza di uno stress biassiale tensile dell'ordine di 2GPa, non ha alcun influsso sul valore di Tc, mentre permette di stabilizzare la formazione di isole di dimensioni minori rispetto a quelle che si avrebbero in assenza di strain. Questo risultato, del tutto originale, può avere importanti risvolti tecnologici legati alla realizzazione di quantum dot depositati direttamente su substrato isolante. Alla luce dei risultati ottenuti dalla caratterizzazione dei substrati, sono state implementate delle procedure di preparazione per il loro utilizzo nelle crescite epitassiali che hanno consentito di ottenere superfici prive di contaminanti, quali carbonio ed ossigeno ([C], [O] <0.1%), non affette dalle modificazioni indotte dal fenomeno dell'agglomerazione e di elevata qualità cristallina. L'ottimizzazione dei parametri di crescita ha permesso di ottenere MQW con interfacce ben definite, come dimostrato da immagini TEM che evidenziano la presenza di interfacce planari ed abrupte su scale <0.5 nm ed in genere la possibilità di depositare strati nanometrici (<3 nm) di ottima qualità. La qualità dei materiali cresciuti sui differenti substrati è dimostrata dal raggiungimento di mobilità molto elevate (= 105 cm2/Vs) in gas bidimensionali di elettroni (2DEG). I risultati delle misure ottiche, nelle frequenze del FIR e del MIR, permettono una perfetta identificazione spettrale delle transizioni intersottobanda all'interno delle QW in banda di conduzione. La larghezza di riga delle transizioni è risultata praticamente indipendente dallo spessore delle buche e pari a 6meV, valore del tutto confrontabile con le larghezze ottenute in campioni cresciuti mediante MBE. Le energie delle ISBT, misurate al variare dello spessore del pozzo e della concentrazione delle barriere, hanno mostrato un ottimo accordo con un modello per la descrizione degli stati elettronici basato su un nuovo approccio tight-binding. L'accordo riscontrato dimostra l'accuratezza del modello tight binding nel determinare gli off-set di banda e gli stati elettronici nelle eterostrutture di tipo Si/SiGe ed inoltre ci ha permesso di quantificare tali off-set al variare della composizione e dello stato di strain delle barriere. Gli off-set stimati, che vanno da circa 150 meV a circa 270 meV, risultano del tutto adeguati per lo sviluppo di un QCL operante nella regione dei THz. In conclusione questa tesi dimostra che il silicio è un sistema materiale altamente promettente per lo sviluppo di un QCL basato su transizioni intersottobanda in banda di conduzione. Il lavoro svolto, inoltre, affronta e risolve alcune questioni tecnologicamente importanti, quali l'ottimizzazione delle procedure di crescita mediante CVD, l'unica tecnica compatibile con la produzione su larga scala, e lo sviluppo di procedure di crescita innovative a partire da nuovi substrati per la microelettronica (lo SGOI e il SSOI), mediante i quali è possibile implementare un'efficace ingegneria delle bande su substrato isolante, riducendo, in tal modo, capacità parassite e perdite di corrente nei dispositivi