Synthesis,conformational analysis and in vivo assays of an anti-cancer vaccine that features an unnatural antigen based on an sp2-iminosugar fragment

The Tn antigen (GalNAc-α-1-O-Thr/Ser) is a well-known tumor-associated carbohydrate determinant. The use of glycopeptides that incorporate this structure has become a significant and promising niche of research owing to their potential use as anticancer vaccines. Herein, the conformational preferences of a glycopeptide with an unnatural Tn antigen, characterized by a threonine decorated with an sp²-iminosugar-type α-GalNAc mimic, have been studied both in solution, by combining NMR spectroscopy and molecular dynamics simulations, and in the solid state bound to an anti-mucin-1 (MUC1) antibody, by X-ray crystallography. The Tn surrogate can mimic the main conformer sampled by the natural antigen in solution and exhibits high affinity towards anti-MUC1 antibodies. Encouraged by these data, a cancer vaccine candidate based on this unnatural glycopeptide and conjugated to the carrier protein Keyhole Limpet Hemocyanin (KLH) has been prepared and tested in mice. Significantly, the experiments in vivo have proved that this vaccine elicits higher levels of specific anti-MUC1 IgG antibodies than the analog that bears the natural Tn antigen and that the elicited antibodies recognize human breast cancer cells with high selectivity. Altogether, we compile evidence to confirm that the presentation of the antigen, both in solution and in the bound state, plays a critical role in the efficacy of the designed cancer vaccines. Moreover, the outcomes derived from this vaccine prove that there is room for exploring further adjustments at the carbohydrate level that could contribute to designing more efficient cancer vaccines.

An anti-cancer vaccine based on an unnatural antigen with an sp²-iminosugar fragment.     

Functionalized carbon dots as sensors for gold nanoparticles in spiked samples: Formation of nanohybrids

This paper reports the synthesis, passivation and functionalization of luminescent carbon dots (CDs) possessing surface thiol ending groups. A simple procedure involving amidation of passivated carbon dots (p-CDs) with cysteamine boosts their photoluminescent properties and enables their use as easily controlled fluorescent nanosensors for determining citrate–gold nanoparticles (AuNPs). The mechanism behind the quenching phenomenon was established from fluorescence measurements at high temperatures and lifetime tests, and found to involve static quenching leading to the formation of CD–AuNP nanohybrids. A method for determining AuNPs in complex matrices was developed and validated by application to spiked drinking water and mussel tissues. The limits of detection and quantitation for AuNPs thus obtained were 0.20 and 0.66 nmol L^–1, respectively.     

Sulfonylcarbamimidic azides from sulfonyl chlorides and 5-aminotetrazole

Treatment of o-nitrobenzenesulfonyl chloride ( 3 ) with 5-aminotetrazole (5-AT) gave [(2-nitrophenyl)-sulfonyl]carbamimidic azide ( 6 ), a ring-opened isomer of the expected N-(1H-tetrazol-5-yl)-2-nitrobenzenesulfonamide ( 4 ). Sulfonylcarbamimidic azide 6 was converted to 2-amino-N-(aminoiminomethyl)benzene-sulfonamide ( 7 ) with ethanolic stannous chloride, and to 3-amino-1,2,4-thiadiazine 1,1-dioxide ( 8 ) with sodium dithionite. Methanesulfonyl chloride ( 9 ) and 5-AT gave 2-(methylsulfonyl)carbamimidic azide ( 10 ), which isomerized to 5-[(methylsulfonyl)amino]-1H-tetrazole ( 11 ) in warm ethanol. Attempted cycloaddition of 2-(phenylsulfonyl)carbamimidic azide ( 13 ) and ethyl vinyl ether led only to alkylated tetrazole products. In addition, other tetrazole-alkylating reactions are described. Isomers produced from these alkylations were differentiated with ¹³C nmr spectroscopy.     

Improved protein identification efficiency by mass spectrometry using N-terminal chemical derivatization of peptides from Angiostrongylus costaricensis, a nematode with unknown genome

Matrix-assisted laser desorption ionization (MALDI), Peptide Mass Fingerprinting (PMF) and MALDI-MS/MS ion search (using MASCOT) have become the preferred methods for high-throughput identification of proteins. Unfortunately, PMF can be ambiguous, mainly when the genome of the organism under investigation is unknown and the quality of spectra generated is poor and does not allow confident identification. The post-source decay (PSD) fragmentation of singly charged tryptic peptide ions generated by MALDI-TOF/TOF typically results in low fragmentation efficiency and/or complex spectra, including backbone fragmentation ions (series b and y), internal fragmentation etc. Interpreting these data either manually and/or using de novo sequencing software can frequently be a challenge. To overcome this limitation when studying the proteome of adult Angiostrongylus costaricensis, a nematode with unknown genome, we have used chemical N-terminal derivatization of the tryptic peptides with 4-sulfophenyl isothiocyanate (SPITC) prior to MALDI-TOF/TOF MS. This methodology has recently been reported to enhance the quality of MALDI-TOF/TOF-PSD data, allowing the obtainment of complete sequence of most of the peptides and thus facilitating de novo peptide sequencing. Our approach, consisting of SPITC derivatization along with manual spectra interpretation and Blast analysis, was able to positively identify 76% of analyzed samples, whereas MASCOT analysis of derivatized samples, MASCOT analysis of nonderivatized samples and PMF of nonderivatized samples yielded only 35, 41 and 12% positive identifications, respectively. Moreover, de novo sequencing of SPITC modified peptides resulted in protein sequences not available in NCBInr database paving the way to the discovery of new protein molecules.     

Effect of Surface Treatment of Titanium Dioxide Nanoparticles on Non-Isothermal Crystallization Behavior,Viscoelastic Transitions and Cold Crystallization of Poly(Ethylene Terephthalate) Nanocomposites

The effect of untreated and tri-n-octylphosphine oxide (TOPO) surface-treated TiO₂ nanoparticles when included as filler in poly(ethylene terephthalate) on its compatibility, non-isothermal crystallization behavior, viscoelastic transitions and cold crystallization has been studied. The effectiveness of the surface treatment has been studied using infrared spectrophotometry (FTIR) and thermogravimetric analysis (TGA). The effect of the untreated and surface-treated nanofiller content in the polymer, added by an extrusion process, on the non-isothermal crystallization has been studied by differential scanning calorimetry (DSC). The influence on the viscoelastic transitions and cold crystallization of PET nanocomposites has been studied through thermomechanical analysis (TMA). The surface treatment and the concentration of nanofiller influence the non-isothermal crystallization behavior, the viscoelastic transitions and the cold crystallization of the PET nanocomposites, enables us to evaluate the compatibility and the level of dispersion/aggregation of the nanofiller in the poly(ethylene terephthalate).     

Isoperimetric inequalities for an ergodic stochastic control problem

In this paper we deal with blow-up solutions to an elliptic equation with a nonlinear gradient term. The problem under consideration can be seen as the ergodic limit of a stochastic control problem with state constraints. It is well known that it has a solution only when a parameter which appears in the equation assumes a particular value known as ergodic constant. For such a constant many properties similar to those of an eigenvalue hold true. We show that a Faber–Krahn inequality can be stated for the ergodic constant and that for the corresponding solution a comparison result in terms of the solution to a symmetrized problem can be proved.     

Accurate mass measurements by Fourier transform mass spectrometry in the study of advanced glycation end products/peptides

The Maillard reaction occurring between sugars and amino groups is important in living systems. When amino groups belonging to protein chains are involved, the Maillard reaction has been invoked as responsible for protein cross-linking and the production of 'toxic' compounds. The reaction leads to the production of a heterogeneous group of substances, usually called advanced glycation end products (AGEs). Classical analytical approaches, such as spectroscopic (ultraviolet, fluorescence) and mass spectrometric (matrix-assisted laser desorption/ionization, liquid chromatography/electrospray ionization mass spectrometry) methods, have shown that the digestion mixture is highly complex. However, there are clear differences between the digestion mixtures of glycated and unglycated human serum albumin (HSA). In the former case, possible glycated peptides belonging to the AGE peptide class may be identified. Tandem mass spectrometric experiments on selected species seemed to be promising as regards structural information, but it was thought of interest to undertake the present investigation, based on liquid chromatography/electrospray ionization Fourier transform mass spectrometry, in order to obtain definitive results on their elemental composition. Using this approach, about 20 glycated peptides were detected and their possible structures were postulated by examining the known sequence of HSA.     

Liquid-crystalline bisadducts of [60]fullerene

A second-generation cyanobiphenyl-based dendrimer was used as a liquid-crystalline promoter to synthesize mesomorphic bisadducts of [60]fullerene. Liquid-crystalline trans-2, trans-3, and equatorial bisadducts were obtained by condensation of the liquid-crystalline promoter, which carries a carboxylic acid function, with the corresponding bisaminofullerene derivatives. A monoadduct of fullerene was also prepared for comparative purposes. All the compounds gave rise to smectic A phases. An additional mesophase, which could not be identified, was observed for the trans-2 derivative. The supramolecular organization of the monoadduct derivative is governed by steric constraints. Indeed, for efficient space filling, adequacy between the cross-sectional areas of fullerene (approximately 100 A(2)) and of the mesogenic groups (approximately 22-25 A(2) per mesogenic group) is required. As a consequence, the monoadduct forms a bilayered smectic A phase. The supramolecular organization of the bisadducts is essentially governed by the nature and structure of the mesogenic groups and dendritic core. Therefore, the bisadducts form monolayered smectic A phases. The title compounds are promising supramolecular materials as they combine the self-organizing behavior of liquid crystals with the properties of fullerene.     

Protein delivery based on uncoated and chitosan-coated mesoporous silicon microparticles

Mesoporous silicon is a biocompatible, biodegradable material that is receiving increased attention for pharmaceutical applications due to its extensive specific surface. This feature enables to load a variety of drugs in mesoporous silicon devices by simple adsorption-based procedures. In this work, we have addressed the fabrication and characterization of two new mesoporous silicon devices prepared by electrochemistry and intended for protein delivery, namely: (i) mesoporous silicon microparticles and (ii) chitosan-coated mesoporous silicon microparticles. Both carriers were investigated for their capacity to load a therapeutic protein (insulin) and a model antigen (bovine serum albumin) by adsorption. Our results show that mesoporous silicon microparticles prepared by electrochemical methods present moderate affinity for insulin and high affinity for albumin. However, mesoporous silicon presents an extensive capacity to load both proteins, leading to systems were protein could represent the major mass fraction of the formulation. The possibility to form a chitosan coating on the microparticles surface was confirmed both qualitatively by atomic force microscopy and quantitatively by a colorimetric method. Mesoporous silicon microparticles with mean pore size of 35 nm released the loaded insulin quickly, but not instantaneously. This profile could be slowed to a certain extent by the chitosan coating modification. With their high protein loading, their capacity to provide a controlled release of insulin over a period of 60-90 min, and the potential mucoadhesive effect of the chitosan coating, these composite devices comprise several features that render them interesting candidates as transmucosal protein delivery systems.