Search

β-Glucosidase (BG) catalyzes the hydrolysis of cellobiose to glucose, a substrate for fermentation to produce the carbon-neutral fuel bioethanol. Enzyme thermal stability and reusability can be improved through immobilization onto insoluble supports. Moreover, nanoscaled matrixes allow for preserving high reaction rates. In this work, BG was physically immobilized onto wrinkled SiO2 nanoparticles (WSNs). The adsorption procedure was tuned by varying the BG:WSNs weight ratio to achieve the maximum controllability and maximize the yield of immobilization, while different times of immobilization were monitored. Results show that a BG:WSNs ratio equal to 1:6 wt/wt provides for the highest colloidal stability, whereas an immobilization time of 24 h results in the highest enzyme loading (135 mg/g of support) corresponding to 80% yield of immobilization. An enzyme corona is formed in 2 h, which gradually disappears as the protein diffuses within the pores. The adsorption into the silica structure causes little change in the protein secondary structure. Furthermore, supported enzyme exhibits a remarkable gain in thermal stability, retaining complete folding up to 90 °C. Catalytic tests assessed that immobilized BG achieves 100% cellobiose conversion. The improved adsorption protocol provides simultaneously high glucose production, enhanced yield of immobilization, and good reusability, resulting in considerable reduction of enzyme waste in the immobilization stage.

The need to protect human and environmental health and avoid the widespread use of substances obtained from nonrenewable sources is steering research toward the discovery and development of new molecules characterized by high biocompatibility and biodegradability. Due to their very widespread use, a class of substances for which this need is particularly urgent is that of surfactants. In this respect, an attractive and promising alternative to commonly used synthetic surfactants is represented by so-called biosurfactants, amphiphiles naturally derived from microorganisms. One of the best-known families of biosurfactants is that of rhamnolipids, which are glycolipids with a headgroup formed by one or two rhamnose units. Great scientific and technological effort has been devoted to optimization of their production processes, as well as their physicochemical characterization. However, a conclusive structure–function relationship is far from being defined. In this review, we aim to move a step forward in this direction, by presenting a comprehensive and unified discussion of physicochemical properties of rhamnolipids as a function of solution conditions and rhamnolipid structure. We also discuss still unresolved issues that deserve further investigation in the future, to allow the replacement of conventional surfactants with rhamnolipids.

The superfamily of vertebrate ribonucleases, a large group of evolutionarily related proteins, continues to provide interesting structural and functional information. In particular, the crystal structure of SS-RNase-2 from Salmo salar (SS2), here presented, has revealed a novel auto-inhibition mechanism that enriches the number of inhibition strategies observed in some members of the family. Within an essentially unmodified RNase folding, the SS2 active site cleft is in part obstructed by the collapse of an extra pentapeptide inserted in the C-terminal region. This unexpected intrusion alters the organization of the catalytic triad by pushing one catalytic histidine off the pocket. Possible mechanisms to remove the active site obstruction have also been studied through the production of two mutants that provide useful information on the functionality of this intriguing version of the ribonuclease superfamily.

The use of technologies based on soft polymer particles represents an effective way to deliver target molecules with a specific function. To design a well-performing delivery system, it is fundamental to rationalize both the aggregation and the structural properties of such particles. In this study, we present the kinetic and structural characterization over time of poly(vinyl alcohol) (PVA) microgels obtained through a salting-out process in the presence of NaCl. We have analyzed how both the polymer and salt concentrations affect the aggregation process. The aggregation rate as well as the morphology and physico-chemical parameters, such as mass and chain density of the microgels, have been determined through static and dynamic light scattering and discussed in the framework of the diffusion-limited and reaction-limited colloid aggregation. Insights into the polymer chain arrangements and their dynamics have been gained by means of small-angle neutron scattering and neutron spin-echo measurements. As a result, it was found that NaCl induces a liquid phase separation in solution with the formation of spherical PVA microaggregates, which grow under a reaction-limited aggregation mechanism. The particles increase their size and compactness over time. Within the aggregate, the polymer chains are locally organized to form randomly oriented lamellae with a thickness of about 60 Å. The internal dynamics is a complex mixture of diffusion, Zimm dynamics, and possibly effects from crowding with the transition to a Rouse-like behavior. The microparticle preparation based on the salting-out process constitutes a novelty, if compared to the methods already existing and based on the use of chemical cross-linkers, and is a cheap and easy protocol that allows tuning both particle size and density by varying the salt concentration.

Extracellular polysaccharides are widely produced by bacteria, yeasts, and algae. These polymers are involved in several biological functions, such as bacteria adhesion to surface and biofilm formation, ion sequestering, protection from desiccation, and cryoprotection. The chemical characterization of these polymers is the starting point for obtaining relationships between their structures and their various functions. While this fundamental correlation is well reported and studied for the proteins, for the polysaccharides, this relationship is less intuitive. In this paper, we elucidate the chemical structure and conformational studies of a mannan exopolysaccharide from the permafrost isolated bacterium Psychrobacter arcticus strain 273-4. The mannan from the cold-adapted bacterium was compared with its dephosphorylated derivative and the commercial product from Saccharomyces cerevisiae. Starting from the chemical structure, we explored a new approach to deepen the study of the structure/activity relationship. A pool of physicochemical techniques, ranging from small-angle neutron scattering (SANS) and dynamic and static light scattering (DLS and SLS, respectively) to circular dichroism (CD) and cryo-transmission electron microscopy (cryo-TEM), have been used. Finally, the ice recrystallization inhibition activity of the polysaccharides was explored. The experimental evidence suggests that the mannan exopolysaccharide from P. arcticus bacterium has an efficient interaction with the water molecules, and it is structurally characterized by rigid-rod regions assuming a 14-helix-type conformation.

Simple Summary The availability of selective, effective, and safe anticancer agents is a major challenge in the field of cancer research. As part of a multidisciplinary research project, in recent years our group has proposed an original class of nanomaterials for the delivery of new anticancer drugs based on ruthenium(III) complexes. In cellular models, these nanosystems have been shown to be effective in counteracting growth and proliferation of human breast cancer cells. Compared to conventional metallochemotherapeutics such as platinum-based agents whose clinical practice is associated with serious undesirable effects, ruthenium complexes share improved biochemical profiles making them more selective towards cancer cells and less cytotoxic to healthy cells. Their combination with biocompatible nanocarriers further enhances these promising features, as here showcased by our research carried out in an animal model which underscores the efficacy and safety in vivo of one of our most promising ruthenium-based nanosystems. Abstract Selectivity and efficacy towards target cancer cells, as well as biocompatibility, are current challenges of advanced chemotherapy powering the discovery of unconventional metal-based drugs and the search for novel therapeutic approaches. Among second-generation metal-based chemotherapeutics, ruthenium complexes have demonstrated promising anticancer activity coupled to minimal toxicity profiles and peculiar biochemical features. In this context, our research group has recently focused on a bioactive Ru(III) complex—named AziRu—incorporated into a suite of ad hoc designed nucleolipid nanosystems to ensure its chemical stability and delivery. Indeed, we proved that the structure and properties of decorated nucleolipids can have a major impact on the anticancer activity of the ruthenium core. Moving in this direction, here we describe a preclinical study performed by a mouse xenograft model of human breast cancer to establish safety and efficacy in vivo of a cationic Ru(III)-based nucleolipid formulation, named HoThyRu/DOTAP, endowed with superior antiproliferative activity. The results show a remarkable reduction in tumour with no evidence of animal suffering. Blood diagnostics, as well as biochemical analysis in both acute and chronic treated animal groups, demonstrate a good tolerability profile at the therapeutic regimen, with 100% of mice survival and no indication of toxicity. In addition, ruthenium plasma concentration analysis and tissue bioaccumulation were determined via appropriate sampling and ICP-MS analysis. Overall, this study supports both the efficacy of our Ru-containing nanosystem versus a human breast cancer model and its safety in vivo through well-tolerated animal biological responses, envisaging a possible forthcoming use in clinical trials.

The integration of nuclear imaging analysis with nanomedicine has tremendously grown and represents a valid and powerful tool for the development and clinical translation of drug delivery systems. Among the various types of nanostructures used as drug carriers, nanovesicles represent intriguing platforms due to their capability to entrap both lipophilic and hydrophilic agents, and their well-known biocompatibility and biodegradability. In this respect, here we present the development of a labelling procedure of POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)-based liposomes incorporating an ad hoc designed lipophilic NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) analogue, derivatized with an oleic acid residue, able to bind the positron emitter gallium-68(III). Based on POPC features, the optimal conditions for liposome labelling were studied with the aim of optimizing the Ga(III) incorporation and obtaining a significant radiochemical yield. The data presented in this work demonstrate the feasibility of the labelling procedure on POPC liposomes co-formulated with the ad hoc designed NOTA analogue. We thus provided a critical insight into the practical aspects of the development of vesicles for theranostic approaches, which in principle can be extended to other nanosystems exploiting a variety of bioconjugation protocols.

In recent years, interest in the class of water-soluble polymers—hydrophobically modified alkali soluble emulsion (HASE)—has increased on a surprising scale. With respect to other associative polymers, they have several advantages in terms of cost and ease of handling and utilization. In addition, unlike the solvent-based formulations, these water-soluble systems do not contain volatile solvents and hence do not contribute to environmental problems. In solutions, HASE polymers form a transient network through molecular associations between the hydrophobic groups. Due to these hydrophobic interactions, they have been increasingly used as rheology modifiers for various applications, such as paints, cosmetics, and personal care and paper coating products. These associative polymers have an architectural richness that allows the fine tuning of several physicochemical properties, and their optimal use requires controlling polymer concentration, molecular weight, size of the hydrophobic groups, and characteristics of the polymeric backbone. We give an overview of several studies on HASE polymers reported in the literature, focusing on the molecular structure, the synthetic methodologies, and—more specifically—on the factors that affect the rheology of their aqueous mixtures. As a general rule for the optimal HASE design and formulative exploitation, we highlight that the hydrophobic hanging groups are responsible for the rheological changes in the liquid phase, while the steric hindrance of the polymeric backbone and of the hydrophobic groups causes the stiffness.

Nanoparticles (NPs) are increasingly exploited as diagnostic and therapeutic devices in medicine. Among them, superparamagnetic nanoparticles (SPIONs) represent very promising tools for magnetic resonance imaging, local heaters for hyperthermia, and nanoplatforms for multimodal imaging and theranostics. However, the use of NPs, including SPIONs, in medicine presents several issues: first, the encounter with the biological world and proteins in particular. Indeed, nanoparticles can suffer from protein adsorption, which can affect NP functionality and biocompatibility. In this respect, we have investigated the interaction of small SPIONs covered by an amphiphilic double layer of oleic acid/oleylamine and 1-octadecanoyl-sn-glycero-3-phosphocholine with two abundant human plasma proteins, human serum albumin (HSA) and human transferrin. By means of spectroscopic and scattering techniques, we analyzed the effect of SPIONs on protein structure and the binding affinities, and only found strong binding in the case of HSA. In no case did SPIONs alter the protein structure significantly. We structurally characterized HSA/SPIONs complexes by means of light and neutron scattering, highlighting the formation of a monolayer of protein molecules on the NP surface. Their interaction with lipid bilayers mimicking biological membranes was investigated by means of neutron reflectivity. We show that HSA/SPIONs do not affect lipid bilayer features and could be further exploited as a nanoplatform for future applications. Overall, our findings point toward a high biocompatibility of phosphocholine-decorated SPIONs and support their use in nanomedicine.

Polyunsaturated omega-3 fatty acid docosahexaenoic acid (DHA) is found in very high concentrations in a few peculiar tissues, suggesting that it must have a specialized role. DHA was proposed to affect the function of the cell membrane and related proteins through an indirect mechanism of action, based on the DHA-phospholipid effects on the lipid bilayer structure. In this respect, most studies have focused on its influence on lipid-rafts, somehow neglecting the analysis of effects on liquid disordered phases that constitute most of the cell membranes, by reporting in these cases only a general fluidifying effect. In this study, by combining neutron reflectivity, cryo-transmission electron microscopy, small angle neutron scattering, dynamic light scattering and electron paramagnetic resonance spectroscopy, we characterize liquid disordered bilayers formed by the naturally abundant 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and different contents of a di-DHA glycero-phosphocholine, 22:6-22:6PC, from both a molecular/microscopic and supramolecular/mesoscopic viewpoint. We show that, below a threshold concentration of about 40% molar percent, incorporation of 22:6-22:6PC in the membrane increases the lipid dynamics slightly but sufficiently to promote the membrane deformation and increase of multilamellarity. Notably, beyond this threshold, 22:6-22:6PC disfavours the formation of lamellar phases, leading to a phase separation consisting mostly of small spherical particles that coexist with a minority portion of a lipid blob with water-filled cavities. Concurrently, from a molecular viewpoint, the polyunsaturated acyl chains tend to fold and expose the termini to the aqueous medium. We propose that this peculiar tendency is a key feature of the DHA-phospholipids making them able to modulate the local morphology of biomembranes. This journal is

International audience; In the search for optimized thrombin binding aptamers (TBAs), we herein describe the synthesis of a library of TBA analogues obtained by end-functionalization with the electron-rich 1,5-dialkoxy naphthalene (DAN) and the electron-deficient 1,8,4,5-naphthalenetetra-carboxylic diimide (NDI) moieties. Indeed, when these G-rich oligonucleotides were folded into the peculiar TBA G-quadruplex (G4) structure, effective donor–acceptor charge transfer interactions between the DAN and NDI residues attached to the extremities of the sequence were induced, providing pseudo-cyclic structures. Alternatively, insertion of NDI groups at both extremities produced TBA analogues stabilized by π–πstacking interactions. All the doubly-modified TBAs were characterized by different biophysical techniques and compared with the analogues carrying only the DAN or NDI residue and unmodified TBA. These modified TBAs exhibited higher nuclease resistance, and their G4 structures were markedly stabilized, as evidenced by increased Tm values compared to TBA. These favorable properties were also associated with improved anticoagulant activity for one DAN/NDI-modified TBA, and for one NDI/NDI-modified TBA. Our results indicated that TBA pseudo-cyclic structuring by ad hoc designed end-functionalization represents an efficient approach to improve the aptamer features, while pre-organizing and stabilizing the G4 structure but allowing sufficient flexibility to the aptamer folding, which is necessary for optimal thrombin recognition.

Formulations for industrial applications are often mixtures of different surfactants and additives, including salts; thus, it is crucial to understand the reciprocal effects of the different components on the properties of the system such as cmc and morphology of the aggregates in solution. In this respect, we have studied the effects of anions occupying different positions within the so-called Hofmeister series, namely Cl−, Br−, SCN−, on the aggregation properties of mixtures containing a cationic and a non-ionic surfactant largely employed in formulation chemistry: dodecyltrimethyl ammonium bromide (DTAB) and pentaethylenglycol mono octyl ether (C8E5). We determined the cmc of the mixtures with different relative content of the two surfactants, as well as of pure DTAB and C8E5 systems, in water and in the presence of salts. Dynamic light scattering measurements were used to evaluate the hydrodynamic size of the aggregates, while small-angle neutron scattering to determine their morphology, as a function of increasing DTAB content, at constant salt concentration. We found no significant effect of salts on the cmc or aggregate dimensions of C8E5. On the other hand, increasing DTAB content, the salt effect is clearly detectable and ion-dependent: salt addition decreases the cmc in the order SCN− ≫ Br− > Cl−; moreover, halides seem to affect structural properties of mixed C8E5-DTAB systems very slightly whereas SCN− leads to a morphological change from a spherical to a cylindrical aggregate shape. Thus, this study well demonstrates how formulation properties can be fine-tuned by salt addition through a proper choice of ions species.

Hypothesis Catanionic vesicles based on large-scale produced surfactants represent a promising platform for the design of innovative, effective and relatively inexpensive nano-vehicles for a variety of actives. Structural, dynamic and functional behavior of these aggregates is finely tuned by the molecular features of their components and can be opportunely tailored for their applications as drug carriers. Experiments Here we investigate the aggregates formed by CTAC and SDS, two of the most diffused surfactants, by means of Dynamic Light Scattering, Small Angle Neutron Scattering and Electron Paramagnetic Resonance spectroscopy (EPR). The exploitation of these aggregates as nano-vehicles is explored using the poorly water-soluble antioxidant trans -resveratrol ( t -RESV), testing t -RESV solubility and antioxidant activity by means of UV, fluorescence spectroscopy and EPR. Findings The presence of a large stability region of catanionic vesicles on the CTAC-rich side of the phase diagram is highlighted and interpreted in terms of the mismatch between the lengths of the surfactant tails and of first reported effects of the chloride counterions. CTAC-SDS vesicles massively solubilize t -RESV, which in catanionic vesicles exerts a potent antioxidant and radical-scavenging activity. This behavior arises from the positioning of the active at the surface of the vesicular aggregates thus being sufficiently exposed to the external medium.

International audience; A small library of cyclic TBA analogues (named cycTBA I-IV), obtained by covalently connecting its 5'- and 3'ends with flexible linkers, has been synthesized with the aim of improving its chemical and enzymatic stability, as well as its anticoagulant properties. Two chemical procedures have been exploited to achieve the desired cyclization, based on the oxime ligation method (providing cycTBA I and II) or on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC) protocols (for cycTBA III and IV), leading to analogues containing circularizing linkers with different chemical nature and length, overall spanning from 22 to 48 atoms. The resulting cyclic TBAs have been characterized using a variety of biophysical methods (UV, CD, gel electrophoresis, SE-HPLC analyses) and then tested for their serum resistance and anticoagulant activity under in vitro experiments. A fine-tuning of the length and flexibility of the linker allowed identifying a cyclic analogue, cycTBA II, with improved anticoagulant activity, associated with a dramatically stabilized G-quadruplex structure (Delta T-m = +17 degrees C) and a 6.6-fold higher enzymatic resistance in serum compared to unmodified TBA.

International audience; With the aim of developing a new approach to obtain improved aptamers, a cyclic thrombin-binding aptamer (TBA) analogue (cycTBA) has been prepared by exploiting a copper(I)-assisted azide-alkyne cycloaddition. The markedly increased serum resistance and exceptional thermal stability of the G-quadruplex versus TBA were associated with halved thrombin inhibition, which suggested that some flexibility in the TBA structure was necessary for protein recognition.

Amine-oxide surfactants have emerged as highly stable, nontoxic, and cost-effective constituents of detergent formulations, specifically as wetting agents and foam boosters. With the aim of enhancing their functional behavior, a new member of this family, N,N-dimethyl-2-propylheptan-1-amine oxide, bearing a branched alkyl tail (C 10 DAO-branched) was synthesized and purified using a simple and easily scalable strategy starting from 2-propylheptan-1-ol. 2D-nuclear magnetic resonance (NMR) and mass spectrometry confirm the obtainment of the desired product in high yield and purity. The protonation behavior of the branched surfactant is not affected by alkyl tail branching, as shown by potentiometric titrations. In contrast, surface activity and aggregation behavior of C 10 DAO-branched is dramatically different from that of the linear analog N,N-dimethyldecyl-1-amine oxide (C 10 DAO-linear), in that it occupies a higher area at the solution interface and aggregates at much higher concentration, forming larger aggregates, as detected using tensiometry and dynamic light scattering (DLS), respectively. Aggregation behavior of C 10 DAO-branched is less sensitive to pH variations. Foaming tests show that C 10 DAO-branched is a more effective foam booster than its linear analog, in both acidic and basic solutions. The experimental results indicate that the branched surfactant can be used in applications that require enhanced and pH-independent surface activity and foamability.

The products of the reaction between cisplatin (CDDP) and the model protein hen egg white lysozyme (HEWL) at 20, 37 and 55 °C in pure water were studied by UV-Vis absorption spectroscopy, intrinsic fluorescence and circular dichroism, dynamic and electrophoretic light scattering and inductively coupled plasma mass spectrometry. X-ray structures were also solved for the adducts formed at 20 and 55 °C. Data demonstrate that high temperature facilitates the formation of CDDP-HEWL adducts, where Pt atoms bind ND1 atom of His15 or NE2 atom of His15 and NH1 atom of Arg14. Our study suggests that high human body temperature (fever) could increase the rate of drug binding to proteins thus enhancing possible toxic side effects related to CDDP administration.

NU172&mdash, a 26-mer oligonucleotide able to bind exosite I of human thrombin and inhibit its activity&mdash, was the first aptamer to reach Phase II clinical studies as an anticoagulant in heart disease treatments. With the aim of favoring its functional duplex-quadruplex conformation and thus improving its enzymatic stability, as well as its thrombin inhibitory activity, herein a focused set of cyclic NU172 analogues&mdash, obtained by connecting its 5&prime, and 3&prime, extremities with flexible linkers&mdash, was synthesized. Two different chemical approaches were exploited in the cyclization procedure, one based on the oxime ligation method and the other on Cu(I)-assisted azide-alkyne cycloaddition (CuAAC), affording NU172 analogues including circularizing linkers with different length and chemical nature. The resulting cyclic NU172 derivatives were characterized using several biophysical techniques (ultraviolet (UV) and circular dichroism (CD) spectroscopies, gel electrophoresis) and then investigated for their serum resistance and anticoagulant activity in vitro. All the cyclic NU172 analogues showed higher thermal stability and nuclease resistance compared to unmodified NU172. These favorable properties were, however, associated with reduced&mdash, even though still significant&mdash, anticoagulant activity, suggesting that the conformational constraints introduced upon cyclization were somehow detrimental for protein recognition. These results provide useful information for the design of improved analogues of NU172 and related duplex-quadruplex structures.

We report here the preparation, physico-chemical characterization, and biological evaluation of a new liposome formulation as a tool for tumor angiogenesis inhibition. Liposomes are loaded with sunitinib, a tyrosine kinase inhibitor, and decorated with cyclo-aminoprolineRGD units (cAmpRGD), efficient and selective ligands for integrin αVβ3. The RGD units play multiple roles since they target the nanovehicles at the integrin αVβ3-overexpressing cells (e.g. activated endothelial cells), favor their active cell internalization, providing drug accumulation in the cytoplasm, and likely take part in the angiogenesis inhibition by interfering in the αVβ3-VEGFR2 cross-talk. Both in vitro and in vivo studies show a better efficacy of this integrated antiangiogenic tool with respect to the free sunitinib and untargeted sunitinib-loaded liposomes. This system could allow a lower administration of the drug and, by increasing the vector specificity, reduce side-effects in a prolonged antiangiogenic therapy.

AS1411 is a nucleolin-binding aptamer which attracted great interest as active targeting ligand for the selective delivery of therapeutic agents to tumour cells. In this work we selected three AS1411 derivatives 5′-conjugated with lipophilic tails and studied their properties in view of their application in liposomial formulations and/or lipid coated-nanoparticles for targeted therapies. The conformational behaviour of these AS1411 analogs has been investigated in comparison with the unmodified aptamer by CD, UV, PAGE, SEC-HPLC, DLS and thioflavin T (ThT) fluorescence assays to get insight in their secondary structure and aggregation properties. This study has been performed in pseudo-physiological buffers mimicking the extra- and intracellular environments, and at different concentrations in the μM range, paying special attention to the effects of the lipophilic tail on the overall aptamer conformation. The 5′-lipidated AS1411 derivatives proved to fold into stable, parallel unimolecular G-quadruplex structures, forming large aggregates, mainly micelles, at conc. >10 μM. Preliminary bioscreenings on selected cancer cells showed that these derivatives are less cytotoxic than AS1411, but maintain a similar biological behaviour. This study demonstrated that lipophilic tails dramatically favour the formation of AS1411 aggregates, however not impairing the formation and thermal stability of its peculiar G4 motifs.

The ion-specific effect on the solubility of macromolecules in water, rationalized through the Hofmeister series, can be used as an effective method to obtain polymer particles with desired microstructural properties thanks to the so-called salting out effect. By choosing the proper salt and optimizing the polymer and the salt concentration, it is possible to tailor the particle structural features like the radius and the degree of compactness and to speed up the aggregation process until complete conversion from free chains to aggregates. Here we rationalize the segregation of poly-ethylene-vinyl alcohol (EVOH) chains in the presence of sodium chloride (NaCl). The effect of polymer and salt concentrations on the kinetics and the thermodynamics of the aggregation process is considered. In particular, we show how such parameters influence the microstructural properties of the particles through a combination of light and neutron scattering techniques as well as microscopy. We found that by increasing sodium chloride concentration it is possible to speed up the aggregation process, obtaining a complete conversion from free chains to aggregates at shorter times, whereas increasing poly-ethylene-vinyl alcohol concentration from 0.70% w/w to 1.30% w/w determines an increase of the microgel size of a two-times factor. Moreover, NaCl concentration directly affects both the packing degree of the polymer chains within the aggregate and the morphology of the polymer particle, leading to a more compact structure at higher NaCl concentrations. Understanding how it is possible to tune both aggregation kinetics and EVOH particle microstructure is fundamental in order to give a general picture which could be broadened to the class of vinyl alcohol-based polymers.

In the search for more efficient anticancer treatments, Ru(III) complexes have attracted much interest among metal-based candidate drugs, showing marked antitumor and antimetastatic activity associated with lower systemic toxicity. Remarkable examples are the Ru(III) complexes NAMI-A and KP1019, which have reached advanced clinical evaluation. In order to improve the in vivo stability of Ru(III)-based drugs, as well as their cellular uptake and effectiveness, a new approach has been proposed by our research group, based on the incorporation of the active, NAMI-A-like Ru(III) complex into highly functionalized nucleolipidic structures, i.e., hybrid molecules containing a nucleoside or nucleotide central core derivatized with a lipid chain, ensuring both efficient protection against extracellular degradation and high cellular internalization of the metal. Aiming at expanding the chemical diversity of available amphiphilic Ru(III) complexes, we here selected a trifunctional α-amino acid to replace the nucleosidic core of previously prepared nucleolipid-based Ru(III) complexes. The amino acidic scaffold, linked to the Ru(III) complex, is decorated with both hydrophilic and lipophilic moieties, conferring high propensity to form stable aggregates in water, which is required to obtain a suitable nanocarrier for the drug delivery. Following this approach, a novel compound, indicated here as compound I, was successfully prepared and characterized, then studied in coformulation with the biocompatible cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP) by dynamic light scattering (DLS), small angle neutron scattering (SANS), and UV–vis analysis. Evaluated in vitro on a panel of human and nonhuman cell lines, it showed good antiproliferative activity on cancer cells, with IC50 values in the μM range, and no relevant cytotoxicity on the healthy cells used as control.

Background Nucleophosmin-1 (NPM1) is an abundant multifunctional protein, implicated in a variety of biological processes and in the pathogenesis of several human malignancies. Its C-terminal domain (CTD) is endowed with a three helix bundle and we demonstrated that several regions within it, associated with acute myeloid leukemia (AML), have a strong tendency to form beta amyloid-like assemblies toxic for cells. The central helix of the bundle (H2) resulted the most amyloidgenic region; here we aim to model the cytoxicity processes of the H2 sequence and getting clues of a potential involvement in toxicity of the interaction between CTDs and cellular membranes. Methods We investigated the interaction of CTD-NPM1 regions with model membranes through fluorescence, SPR, CD and ESR spectroscopies and the localization of NPM1 by immune-fluorescence in leukemic cells. Results Our findings indicate that investigated regions are able to interact with membranes with different mechanisms and outlined the importance of the presence of cholesterol. Conclusions H2 showed a preference of interaction with membrane containing cholesterol determining a sensitive fluidification of the bilayer, while N-term H2 causes a stiffening of central and outer regions of the lipid system. Noticeably, NPM1 mut A demonstrated to thicken at the plasma membrane, differently from wt. These findings were corroborated by diverse mechanisms of interaction of CTDs toward membrane models in vitro. General significance This study suggests that the direct interaction of several regions of NPM1CTD with cellular membranes could be implicated in diseases where NPM1 is mutated and/or where its overexpression is cytoxic.

The characterization of the interactions between biological macromolecules (proteins and nucleic acids) and metal-based drugs is a fundamental prerequisite for understanding their mechanisms of action. X-ray crystallography enables the structural analysis of such complexes with atomic level detail. However, this approach requires the preparation of highly diffracting single crystals, the measurement of diffraction patterns and the structural analysis and interpretation of macromolecule-metal interactions from electron density maps. In this review, we describe principles and methods used to grow and optimize crystals of protein-metallodrug adducts, to determine metal binding sites and to assign and validate metal ligands. Examples from the literature and experience in our own laboratory are provided and key challenges are described, notably crystallization and molecular model refinement against the X-ray diffraction data.

International audience; Progress in understanding and treatment of thrombotic diseases requires new effective methods for the easy, rapid, and reversible control of coagulation processes. In this framework, the use of aptamers, and particularly of the thrombin binding aptamer (TBA), has aroused strong interest, due to its enormous therapeutic potential, associated with a large number of possible applications in biotechnological and bioanalytical fields. Here, we describe a new TBA analogue (named tris-mTBA), carrying three different pendant groups: a dansyl residue at the 3 '- and a beta-cyclodextrin moiety at the 5 '-end-providing a host-guest system which exhibits a marked fluorescence enhancement upon TBA G-quadruplex folding-and a biotin tag, allowing the attachment of the aptamer onto biocompatible streptavidin-coated silica nanoparticles (NPs) of 50 nm hydrodynamic diameter (Sicastar). The use of nanoparticles for the in vivo delivery of TBA, expected to induce per se increased nuclease resistance and improved pharmacokinetic properties of this oligonucleotide, offers as an additional advantage the possibility to exploit multivalency effects, due to the presence of multiple copies of TBA on a single scaffold. In addition, the selected fluorescent system allows monitoring both the presence of TBA on the functionalized NPs and its correct folding upon immobilization, also conferring enhanced enzymatic resistance and bioactivity. The anticoagulant activity of the new tris-mTBA, free or conjugated to Sicastar NPs, was evaluated by dynamic light scattering experiments. Highly effective and reversible inhibition of thrombin activity toward fibrinogen was found for the free tris-mTBA and especially for the tris-mTBA-conjugated NPs, demonstrating great potential for the biomedical control of blood clotting.

Recently, mixed duplex/quadruplex oligonucleotides have attracted great interest for use as biomedical aptamers. In the case of anti-thrombin aptamers, the addition of duplex-forming sequences to a G-quadruplex module identical or very similar to the best-known G-quadruplex of the Thrombin Binding Aptamer (HD1) results in new or improved biological properties, such as higher activity or different recognition properties with respect to HD1. Remarkably, this bimodular fold was hypothesized, based on its sequence, for the only anti-thrombin aptamer in advanced clinical trial, NU172. Whereas cation modulation of G-quadruplex conformation and stability is well characterized, only few data from similar analysis on duplex/quadruplex oligonucleotides exist. Here we have performed a characterization of structure and stability of four different duplex/quadruplex anti-thrombin aptamers, including NU172, in the presence of different cations and in physiological-mimicking conditions in comparison to HD1, by means of spectroscopic techniques (UV and circular dichroism) and differential scanning calorimetry. Our data show a strong reciprocal influence of each domain on the stability of the other and in particular suggest a stabilizing effect of the duplex region in the presence of solutions mimicking the physiological conditions, strengthening the idea that bimodular aptamers present better therapeutic potentialities than those containing a single G-quadruplex domain.

Aptamers directed against human thrombin can selectively bind to two different exosites on the protein surface. The simultaneous use of two DNA aptamers, HD1 and HD22, directed to exosite I and exosite II respectively, is a very powerful approach to exploit their combined affinity. Indeed, strategies to link HD1 and HD22 together have been proposed in order to create a single bivalent molecule with an enhanced ability to control thrombin activity. In this work, the crystal structures of two ternary complexes, in which thrombin is sandwiched between two DNA aptamers, are presented and discussed. The structures shed light on the cross talk between the two exosites. The through-bond effects are particularly evident at exosite II, with net consequences on the HD22 structure. Moreover, thermodynamic data on the binding of the two aptamers are also reported and analyzed.

Locked nucleic acids (LNAs) are formed by bicyclic ribonucleotides where the O2′ and C4′ atoms are linked through a methylene bridge and the sugar is blocked in a 3′-endoconformation. They represent a promising tool for therapeutic and diagnostic applications and are characterized by higher thermal stability and nuclease resistance with respect to their natural counterparts. However, structural descriptions of LNA-containing quadruplexes are rather limited, since few NMR models have been reported in the literature. Here, the first crystallographically derived model of an all-LNA-substituted quadruplex-forming sequence 5′-TGGGT-3′ is presented refined at 1.7 Å resolution. This high-resolution crystallographic analysis reveals a regular parallel G-quadruplex arrangement terminating in a well defined thymine tetrad at the 3′-end. The detailed picture of the hydration pattern reveals LNA-specific features in the solvent distribution. Interestingly, two closely packed quadruplexes are present in the asymmetric unit. They face one another with their 3′-ends giving rise to a compact higher-order structure. This new assembly suggests a possible way in which sequential quadruplexes can be disposed in the crowded cell environment. Furthermore, as the formation of ordered structures by molecular self-assembly is an effective strategy to obtain nanostructures, this study could open the way to the design of a new class of LNA-based building blocks for nanotechnology.

Bovine seminal ribonuclease (BS-RNase) acquires an interesting anti-tumor activity associated with the swapping on the N-terminal. The first direct experimental evidence on the formation of a C-terminal swapped dimer (C-dimer) obtained from the monomeric derivative of BS-RNase, although under non-native conditions, is here reported. The X-ray model of this dimer reveals a quaternary structure different from that of the C-dimer of RNase A, due to the presence of three mutations in the hinge peptide 111–116. The mutations increase the hinge peptide flexibility and decrease the stability of the C-dimer against dissociation. The biological implications of the structural data are also discussed.

Chemical modifications of protein crystals may be achieved via soaking of reactants from their precipitating solution, through the solvent channel, into the protein matrix. We describe a Raman microscopy approach to follow mercury insertion into cysteine pairs within protein single crystals, via soaking in an aqueous Hg2+ solution. The method has been developed using bovine insulin as the model system. Applying an efficient mercuration protocol, consisting of a first step of disulphide bridge TCEP-induced reduction within the crystal, followed by overnight reaction with a HgCl2 solution, we obtained Hg-derivative crystals. Raman spectra collected on these derivative crystals, kept in the mother liquor, reveal a characteristic Raman band at 335 cm−1, which has been assigned to a –S–Hg–S– bridge. The analysis provides Raman-based markers of mercury binding to cysteines, and thus of mercury intoxication.

We report here on an X-ray crystallographic and molecular modeling investigation into the complex 3' interface formed between putative parallel stranded G-quadruplexes and a duplex DNA sequence constructed from the human telomeric repeat sequence TTAGGG. Our crystallographic approach provides a detailed snapshot of a telomeric 3' quadruplex-duplex junction: a junction that appears to have the potential to form a unique molecular target for small molecule binding and interference with telomere-related functions. This unique target is particularly relevant as current high-affinity compounds that bind putative G-quadruplex forming sequences only rarely have a high degree of selectivity for a particular quadruplex. Here DNA junctions were assembled using different putative quadruplex-forming scaffolds linked at the 3' end to a telomeric duplex sequence and annealed to a complementary strand. We successfully generated a series of G-quadruplex-duplex containing crystals, both alone and in the presence of ligands. The structures demonstrate the formation of a parallel folded G-quadruplex and a B-form duplex DNA stacked coaxially. Most strikingly, structural data reveals the consistent formation of a TAT triad platform between the two motifs. This triad allows for a continuous stack of bases to link the quadruplex motif with the duplex region. For these crystal structures formed in the absence of ligands, the TAT triad interface occludes ligand binding at the 3' quadruplex-duplex interface, in agreement with in silico docking predictions. However, with the rearrangement of a single nucleotide, a stable pocket can be produced, thus providing an opportunity for the binding of selective molecules at the interface.

Literature studies carried out by mass spectrometry and X-ray crystallography have demonstrated that cisplatin is able to bind proteins mainly close to Met, His, and free Cys side chains. To identify possible alternative modes of cisplatin binding to proteins at the molecular level, here we have solved the high-resolution X-ray structure of the adduct formed in the reaction between the drug and the model protein thaumatin, which does not contain any His and free Cys residues and possesses just one buried Met. Our data reveal unexpected cisplatin binding sites on the protein surface that could have general significance: cisplatin fragments -[Pt(NH3)2Cl](+), -[Pt(NH3)Cl2], and -[Pt(NH3)2(OH2)](2+) bind to a protein N-terminus and close to Lys and Glu side chains.

γ-glutamyltranspeptidases (γ-GTs) are ubiquitous enzymes that catalyze the hydrolysis of γ-glutamyl bonds in glutathione and glutamine and the transfer of the released γ-glutamyl group to amino acids or short peptides. These enzymes are generally synthesized as precursor proteins, which undergo an intra-molecular autocatalytic cleavage yielding a large and a small subunit. In this study, circular dichroism and intrinsic fluorescence measurements have been used to investigate the structural features and the temperature- and guanidinium hydrochloride (GdnHCl)-induced unfolding of the mature form of the γ-GT from Geobacillus thermodenitrificans (GthGT) and that of its T353A mutant, which represents a mimic of the precursor protein. Data indicate that a) the mutant and the mature GthGT have a different secondary structure content and a slightly different exposure of hydrophobic regions, b) the thermal unfolding processes of both GthGT forms occur through a three-state model, characterized by a stable intermediate species, whereas chemical denaturations proceed through a single transition, c) both GthGT forms exhibit remarkable stability against temperature, but they do not display a strong resistance to the denaturing action of GdnHCl. These findings suggest that electrostatic interactions significantly contribute to the protein stability and that both the precursor and the mature form of GthGT assume compact and stable conformations to resist to the extreme temperatures where G. thermodenidrificans lives. Owing to its thermostability and unique catalytic properties, GthGT is an excellent candidate to be used as a glutaminase in food industry.

Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human ?-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5'-5' polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin-mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin-aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.

Two novel human antitumor immunoconjugates, engineered by fusion of a single-chain antibody fragment against human ErbB2 receptor, termed Erbicin, with either a human RNase or the Fc region of a human IgG1, are selectively cytotoxic for ErbB2-positive cancer cells in vitro and in vivo. These Erbicin-derived immunoagents (EDIAs) do not show the most negative properties of Herceptin, the only humanized mAb against ErbB2 used in the therapy of breast carcinoma: cardiotoxicity and the inability to act on resistant tumors. These differences are probably attributable to the different ErbB2 epitopes recognized by EDIAs and Herceptin, respectively, as we have previously reported that they induce different signaling mechanisms that control tumor and cardiac cell viability. Thus, to accurately identify the novel epitope recognized by EDIAs, three independent and complementary methodologies were used. They gave coherent results, which are reported here: EDIAs bind to a different ErbB2 epitope than Herceptin and the other human/humanized antibodies against ErbB2 reported so far. The epitope has been successfully located in region 122–195 of extracellular domain I. These findings could lead to the identification of novel epitopes on ErbB2 that could be used as potential therapeutic targets to mitigate anti-ErbB2-associated cardiotoxicity and eventually overcome resistance.

The Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph) produces a cold-active iron superoxide dismutase (SOD). PhSOD is a homodimeric enzyme, that displays a high catalytic activity even at low temperature. Using hanging-drop vapour-diffusion technique, PhSOD has been successfully crystallized in two different crystal forms. Both crystal forms are monoclinic with space group P2(1) and diffract to 2.1 A resolution. Form I has unit-cell parameters a=45.49A b=103.63A c=50.37A beta=108.2 degrees and contains a homodimer in the asymmetric unit. Form II has unit-cell parameters a=50.48A b=103.78A c=90.25A beta=103.8 degrees and an asymmetric unit containing two PhSOD homodimers. Structure determination has been achieved using molecular replacement. The crystallographic study of this cold-adapted enzyme could contribute to the understanding of the molecular mechanisms of cold-adaptation and of the high catalytic efficiency at low temperature.

Mixed duplex/quadruplex oligonucleotides have attracted great interest as therapeutic targets as well as effective biomedical aptamers. In the case of thrombin-binding aptamer (TBA), the addition of a duplex motif to the G-quadruplex module improves the aptamer resistance to biodegradation and the affinity for thrombin. In particular, the mixed oligonucleotide RE31 is significantly more effective than TBA in anticoagulation experiments and shows a slower disappearance rate in human plasma and blood. In the crystal structure of the complex with thrombin, RE31 adopts an elongated structure in which the duplex and quadruplex regions are perfectly stacked on top of each other, firmly connected by a well-structured junction. The lock-and-key shape complementarity between the TT loops of the G-quadruplex and the protein exosite I gives rise to the basic interaction that stabilizes the complex. However, our data suggest that the duplex motif may have an active role in determining the greater anti-thrombin activity in biological fluids with respect to TBA. This work gives new information on mixed oligonucleotides and highlights the importance of structural data on duplex/quadruplex junctions, which appear to be varied, unpredictable, and fundamental in determining the aptamer functional properties.

Comment on Time-dependent, protein-directed growth of gold nanoparticles within a single crystal of lysozyme.

Among Fe/Mn superoxide dismutases (SODs) a very peculiar sub-class is that of cambialistic SODs. These proteins are active with either Fe or Mn in the active site, in contrast with the other SODs that are strictly metal-specific. Here we report the metal-dependent regulation of the activity and the crystallographic structure of the cambialistic SODs from the dental pathogen Streptococcus mutans (SmSOD) and the food-industry bacterium Streptococcus thermophilus (StSOD). The two enzymes share a high sequence identity (86.2%) and present very similar three-dimensional structures. A detailed comparison with the other cambialistic SODs, found in the Protein Data Bank, allowed the identification of two sub-groups of cambialistic enzymes, the Fe-like and the Mn-like. In particular, SmSOD and StSOD were classified as belonging to the Mn-like sub-group; this assignment was in good agreement with the activity data, showing a significantly higher catalysis in Mn-bound forms of SmSOD and StSOD with respect to their Fe-forms. However, in spite of a very similar Mn-dependent activity, SmSOD and StSOD display a consistently different Fe-dependent activity, SmSOD being three-times less efficient than StSOD in the Fe-bound form. The analysis of the X-ray structures suggests that this difference could be related to the effect of a fraction of enzyme molecules possessing an atypical hexa-coordinated iron ion in the active site of SmSOD. These new structural data provide deeper insights into the family of cambialistic SODs.

The reaction between cis-diamminedichloroplatinum(II) (CDDP), cisplatin, a common anticancer drug, and bovine pancreatic ribonuclease (RNase A), induces extensive protein aggregation, leading to the formation of one dimer, one trimer and higher oligomers whose yields depend on cisplatin/protein ratio. Structural and functional properties of the purified platinated species, together with their spontaneous dissociation and thermally induced denaturation, have been characterized. Platinated species preserve a significant, although reduced, ribonuclease activity. The high resistance of the dimers against dissociation and the different thermal unfolding profiles suggest a quaternary structure different from those of the well-known swapped dimers of RNase A.

The structure and stability of the adduct formed in the reaction between Auoxo3, a dinuclear gold(III) compound, and the model protein hen egg white lysozyme (HEWL) are investigated by X-ray crystallography, UV-Vis absorption spectroscopy and circular dichroism (CD). It is found that Auoxo3 breaks down completely, undergoes reduction and produces reactive gold(I) species able to bind the protein and form stable derivatives. The behaviour of Auoxo3 is compared with that of two analogous gold(III) complexes previously studied: a few significant differences are highlighted. The general implications of these new results for the mode of action of cytotoxic gold complexes are discussed.

The adducts formed between trans-(dimethylamino)(methylamino)dichloridoplatinum(II), [t-PtCl2(dma)(ma)], and two model proteins, i.e., hen egg white lysozyme and bovine pancreatic ribonuclease, were independently characterized by X-ray crystallography and electrospray ionization mass spectrometry. In these adducts, the Pt(II) center, upon chloride release, coordinates either to histidine or aspartic acid residues while both alkylamino ligands remain bound to the metal. Comparison with the cisplatin derivatives of the same proteins highlights for [t-PtCl2(dma)(ma)] a kind of biomolecular metalation remarkably different from that of cisplatin.