Development of novel maleimide reagents for protein modification

Chemical methods for protein modification are of utmost importance as they allow the mimicking of post-translational modifications that occur in living cells. In addition, they allow exogenous modifications that are the basis for bioconjugation, protein visualisation (fluorescent tagging) and immobilisation (biotin labelling) and many applications in therapeutics. Bromomaleimides are the first of a new class of reagents that could be efficiently used for the highly selective and reversible modification of cysteine and for the bridging of disulfide bonds in proteins. Aiming to prove that these transformations are not restricted to bromomaleimides, the present work presents a library of novel analogues, bearing different leaving groups on the double bond. By controlling the chemistry of this class of compounds we were able to tune properties such as thiol selectivity, reactivity, water-solubility and cross reactivity with reducing agents. The utility of the novel monosubstitued analogues as protein labelling reagents was shown using a single cysteine mutant of protein Grb2 (L111C) as a model system and a kinetic study was designed with the aim to quantify the difference in reactivity of the various selected analogues. The disubstitued analogues were tested as disulfide bridging reagents using Somatostatin, a 14-aminoacid peptide containing a disulfide bridge. Phenoxymaleimides were shown to be less reactive and to display a different reactivity compared to bromomaleimides. They react with reduced disulfides to form a succinimide bridged product. The successful use of phenoxymaleimides in bridging disulfides in peptides and proteins is described. This strategy is fast and efficient and was optimised as to reduce the risk of disulfide scrambling, protein aggregation and loss of activity, which are the common problems associated with disulfide modification. Also, a novel one-pot protocol for the differentially labelling of disulfides in both peptides and proteins was developed. Several dual modified Somatostatin conjugates were prepared using this strategy

Functionalization of a layered oxide with organic moieties: towards hybrid proton conductors

The design of innovative proton conductors for intermediate-temperature fuel cells, closing the gap between PEMFC and SOFC, is a forefront research theme in materials chemistry. [1] Layered perovskites with the Dion-Jacobson structure (ALaNb2O7) have bidimensional lanthanum niobate sheets, separated by a layer of A+ cations. These can be substituted by a variety of molecules with soft chemistry, to yield inorganic-organic hybrids. In particular, the intercalation of amines, alcohols, carboxylic or phosphonic acids, and their covalent binding to the sheets has been demonstrated recently. [2-4]We present preliminary results on the intercalation and covalent bonding of different organic molecules, in order to develop hybrid proton conductors for use in intermediate temperature fuel cells. Smaller molecules (such as alcohols) are intercalated to expand the interlayer space, to form intermediates for the further binding of proton carriers such as imidazoles or sulfonates.The intercalation process is investigated by XRD (to measure the interlayer distance) and TGA (to determine the weight loss upon thermal decomposition). NMR is applied to confirm the covalent bonding between the organic and oxide parts. The intercalation behavior of different functional groups is explained in terms of van der Waals and/or hydrogen bonding between organic chains. The interplay of theory (ab initio and periodic DFT) and experiment allowed us to elucidate the 1H and 13C-NMR spectra, and to investigate the nature of interaction (i.e. ionic or covalent bond) of the organic chains with the interlayer surface

Drived diffusion of vector fields

A model for the diffusion of vector fields driven by external forces is proposed. Using the renormalization group and the $\epsilon$-expansion, the dynamical critical properties of the model with gaussian noise for dimensions below the critical dimension are investigated and new transport universality classes are obtained.Comment: 11 pages, title changed, anisotropic diffusion further discussed and emphasize

Toward a new hybrid proton conductor: lanthanum niobate layered perovskites as a source of tailorable surfaces

The modification of metal oxide surfaces with organic moieties has been widely studied as a method of preparing organic-inorganic hybrid materials for various applications. Among inorganic oxides, the ion-exchangeable layered perovskites [1], materials composed by perovskite-like slabs and intercalated cations, stimulated authors\u2019 interest in reason of some encouraging electronic and reactive properties. In particular it is well known that the interlayer surface of such materials in their protonated form can be easily functionalized with organic groups (such as alcohols [2-3] or organophosphonic acids [4]) thus allowing the production of stable hybrid materials with new electronic and reactive features. As a first step to design a new inorganic-organic hybrid proton conductor, a comprehensive theoretical investigation of the MLaNb2O7 (M=H, Li, Na, K, Rb and Cs) series of ion-exchangeable layered perovskite is presented. In particular, their structural and electronic properties have been investigated by periodic calculations in the framework of DFT. A general very good agreement with the available experimental data has been found. The protonated compound (HLaNb2O7) has been then functionalized with imidazole trying two different settings: in the first arrangement the molecule is adsorbed on the layered oxide exposing the interlayer surface, in the second the organic moiety is just put between two perovskites slabs. This latter model, including the effect of the confinement, allowed to better reproduce the experimental structural XRD data and 13C-NMR measurements of the hybrid system. [1] Schaak, R. E. and Mallouk T. E., Chem. Mat. 2002, 14, 1455-1471. [2] Takahashi S. et al., Inorg. Chem. 1995, 34, 5065-5069. [3] Suzuki H. et al., Chem. Mater. 2003, 15, 636-641. [4] Shimada, A. et al., Chem. Mat. 2009, 21, 4155-4162

Chemical structure of methylmethacrylate-2-[2′,3′,5′-triiodobenzoyl]oxoethyl methacrylate copolymer, radio-opacity, in vitro and in vivo biocompatibility

The properties of copolymers (physical, chemical, biocompatibility, etc.) depend on their chemical structure and microstructural characteristics. We have prepared radio-opaque polymers based on the copolymers of methyl methacrylate (MMA) and 2-[2′,3′,5′-triiodobenzoyl]oxoethyl methacrylate (TIBOM). The copolymerization reaction between TIBOM and MMA showed that the reactivity ratios were r1 = 0.00029 and r2 = 1.2146. The composition diagram is typical for a practically non-homopolymerizable monomer (TIBOM) and a very reactive monomer (MMA). The copolymers were analyzed on an X-ray microcomputed tomograph and they proved to be radio-opaque even at low concentrations of TIBOM. The biocompatibility was tested both in vitro (with J774.2 macrophage and SaOS-2 osteoblast like cells) and in vivo in the rat. These materials were found to be non-toxic and were well tolerated by the organism. These combined results led to the suggestion that this type of polymer could be used as dental or bone cements in place of barium or zirconium particles, which are usually added to provide X-ray opacity

Diagnostic performance and reference values of novel biomarkers of paediatric heart failure

Objective: Biomarkers play a pivotal role in heart failure (HF) management. Reference values and insights from studies in adults cannot be extrapolated to the paediatric population due to important differences in pathophysiology and compensatory reserve. We assessed the diagnostic utility of four novel biomarkers in paediatric HF. Methods: Midregional (MR) pro-atrial natriuretic peptide (proANP), soluble ST2 (sST2), growth differentiation factor-15 (GDF-15), MR-pro-adrenomedullin (proADM) and N-terminal pro-B natriuretic peptide (NT-proBNP) were measured in 114 patients and 89 controls. HF was defined as the presence of HF symptoms and/or abnormal systolic ventricular function. Receiver-operating characteristics were plotted, and the area under the curve (AUC) was measured. This was repeated for subgroups with cardiomyopathy and congenital heart disease (CHD). Ventricular systolic function was measured by magnetic resonance or echocardiography. Reference values were calculated according to the current guidelines. Results: The AUC for diagnosing HF was 0.76 for MR-proANP (CI 0.70 to 0.84) and 0.82 for NT-proBNP (CI 0.75 to 0.88). These parameters performed similarly in the subgroups with CHD and cardiomyopathy. By contrast, MR-proADM, GDF-15 and sST2 performed poorly. When used in conjunction with NT-proBNP, no parameter added significantly to its diagnostic accuracy. NT-proBNP, MR-proANP, GDF-15 and sST2 could accurately discriminate between patients with preserved and patients with poor functional status. In a subset of patients with dilated cardiomyopathy, NT-proBNP, MR-proANP, MR-proADM and GDF-15 were associated with poor LV function. Conclusions: MR-proANP could accurately detect HF in children and adolescents. Its diagnostic performance was comparable with that of NT-proBNP, regardless of the underlying condition. Reference values are presented

Coulomb Effects on Electromagnetic Pair Production in Ultrarelativistic Heavy-Ion Collisions

We discuss the implications of the eikonal amplitude on the pair production probability in ultrarelativistic heavy-ion transits. In this context the Weizs\"acker-Williams method is shown to be exact in the ultrarelativistic limit, irrespective of the produced particles' mass. A new equivalent single-photon distribution is derived which correctly accounts for the Coulomb distortions. As an immediate application, consequences for unitarity violation in photo-dissociation processes in peripheral heavy-ion encounters are discussed.Comment: 13 pages, 4 .eps figure

Soft capacitor fibers using conductive polymers for electronic textiles

A novel, highly flexible, conductive polymer-based fiber with high electric capacitance is reported. In its crossection the fiber features a periodic sequence of hundreds of conductive and isolating plastic layers positioned around metallic electrodes. The fiber is fabricated using fiber drawing method, where a multi-material macroscopic preform is drawn into a sub-millimeter capacitor fiber in a single fabrication step. Several kilometres of fibers can be obtained from a single preform with fiber diameters ranging between 500um -1000um. A typical measured capacitance of our fibers is 60-100 nF/m and it is independent of the fiber diameter. For comparison, a coaxial cable of the comparable dimensions would have only ~0.06nF/m capacitance. Analysis of the fiber frequency response shows that in its simplest interrogation mode the capacitor fiber has a transverse resistance of 5 kOhm/L, which is inversely proportional to the fiber length L and is independent of the fiber diameter. Softness of the fiber materials, absence of liquid electrolyte in the fiber structure, ease of scalability to large production volumes, and high capacitance of our fibers make them interesting for various smart textile applications ranging from distributed sensing to energy storage

Aryloxymaleimides for cysteine modification, disulfide bridging and the dual functionalization of disulfide bonds

Tuning the properties of maleimide reagents holds significant promise in expanding the toolbox of available methods for bioconjugation. Herein we describe aryloxymaleimides which represent 'next generation maleimides' of attenuated reactivity, and demonstrate their ability to enable new methods for protein modification at disulfide bonds

Sustained Effects of Interleukin-1 Receptor Antagonist Treatment in Type 2 Diabetes

Objective: Interleukin (IL)-1 impairs insulin secretion and induces beta-cell apoptosis. Pancreatic beta-cell IL-1 expression is increased and interleukin-1-receptor antagonist (IL-1Ra) expression reduced in patients with type 2 diabetes mellitus. Treatment with recombinant IL-1Ra improves glycemia and beta-cell function and reduces inflammatory markers in patients with type 2 diabetes mellitus. Here we investigated the durability of these responses. Research Design and Methods: Among 70 ambulatory patients with type 2 diabetes and A1C and body mass index higher than 7.5% and 27, respectively, randomly assigned to receive 13 weeks of anakinra, a recombinant human IL-1Ra, or placebo, 67 completed treatment and were included in this double-blinded 39 week follow-up study. Primary outcome was change in betacell function following anakinra withdrawal. Analysis was done by intention-to-treat. Results: Thirty-nine weeks following anakinra withdrawal the proinsulin to insulin (PI/I) ratio but not stimulated C-peptide remained improved by -0.07 (95% CI -0.14 to -0.02, P=0.011) compared to placebo treated patients. Interestingly, a subgroup characterized by genetically determined low baseline IL-1Ra serum levels, maintained the improved stimulated C-peptide obtained by 13 weeks of IL-1Ra treatment. Reductions of C-reactive protein (-3.2 mg/l [95% CI -6.2 to -1.1, P=0.014]) and of IL-6 (-1.4 ng/l [95% CI -2.6 to -0.3, P=0.036]) were maintained until end of study. Conclusions: IL-1 blockade with anakinra induces improvement of the PI/I ratio and in markers of systemic inflammation lasting 39 weeks following treatment withdrawal