A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation

We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization.     

Polyacrylonitrile molecular weight effect on the structural and magnetic properties of metal–carbon nanomaterial

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Comparative Study of Natural Terpenoid Precursors in Reactive Plasmas for Thin Film Deposition

If plasma polymer thin films are to be synthesised from sustainable and natural precursors of chemically heterogeneous composition, it is important to understand the extent to which this composition influences the mechanism of polymerisation. To this end, a well-studied monoterpene alcohol, terpinen-4-ol, has been targeted for a comparative study with the naturally occurring mix of terpenes (viz. Melaleuca alternifolia oil) from which it is commonly distilled. Positive ion mode mass spectra of both terpinen-4-ol and M. alternifolia oil showed a decrease in disparities between the type and abundance of cationic species formed in their respective plasma environments as applied plasma power was increased. Supplementary biological assay revealed the antibacterial action of both terpinen-4-ol and M. alternifolia derived coatings with respect to S. aureus bacteria, whilst cytocompatibility was demonstrated by comparable eukaryotic cell adhesion to both coatings. Elucidating the processes occurring within the reactive plasmas can enhance the economics of plasma polymer deposition by permitting use of the minimum power, time and precursor pre-processing required to control the extent of monomer fragmentation and fabricate a film of the desired thickness and functionality.     

Structure-fluorescence contrast relationship in cyanine DNA intercalators: toward rational dye design

The fluorescence enhancement mechanisms of a series of DNA stains of the oxazole yellow (YO) family have been investigated in detail using steady-state and ultrafast time-resolved fluorescence spectroscopy. The strong increase in the fluorescence quantum yield of these dyes upon DNA binding is shown to originate from the inhibition of two distinct processes: 1) isomerisation through large-amplitude motion that non-radiatively deactivates the excited state within a few picoseconds and 2) formation of weakly emitting H-dimers. As the H-dimers are not totally non-fluorescent, their formation is less efficient than isomerisation as a fluorescent contrast mechanism. The propensity of the dyes to form H-dimers and thus to reduce their fluorescence contrast upon DNA binding is shown to depend on several of their structural parameters, such as their monomeric (YO) or homodimeric (YOYO) nature, their substitution and their electric charge. Moreover, these parameters also have a substantial influence on the affinity of the dyes for DNA and on the ensuing sensitivity for DNA detection. The results give new insight into the development and optimisation of fluorescent DNA probes with the highest contrast.     

In vitro evaluation of cytotoxic and mutagenic activity of avarol

The cytotoxicity of avarol, a main secondary metabolite of the Mediterranean sponge Dysidea avara, was in vitro screened by MTT assay against four human tumour cell lines. The colon HT-29 tumour cells practically showed to be the only sensitive ones towards this organic compound. No toxicity was found against the fetal lung fibroblast MRC-5 cells at the concentrations tested. In comparison with doxorubicin, used as a positive control, avarol actually exhibited at least 588-fold less toxicity towards normal MRC-5 cells. Finally, comet assay indicated that DNA fragmentation was almost fivefold higher upon the treatment with doxorubicin, compared to avarol. The obtained results have actually confirmed that avarol scaffold may contribute to development of new cytostatics inspired by nature.     

A Preparative Method for Crosslinking Proteins to DNA in Nuclei by Single-pulse UV Laser Irradiation

Abstract— Photocrosslinking of proteins to DNA by single-pulse UV laser has been used only in analytical experiments, carried out with reconstituted complexes of a single DNA binding protein and a labeled target sequence. Here we propose a large-scale technique for irradiation of nuclei, generating preparative quantities of covalently linked protein-DNA complexes for further analysis of the partner molecules. The use of a flow cuvette allows a milligram of DNA in either nuclei or chromatin to be irradiated by a single pulse within few minutes. The efficiency of crosslinking varies from 6 to 12% of the total nuclear proteins. The presence of histones and other chromosomal proteins in the crosslinked protein-DNA complexes was demonstrated by using specific antibodies. The irradiation procedure can be fully automated using a microcomputer.     

RAFT synthesis of thioether-based,AB diblock copolymer nanocarriers for reactive oxygen species–triggered release

A well-defined AB diblock copolymer of 2-vinyl-4,4-dimethylazlactone (VDA) and N,N-dimethylacrylamide (DMA) was generated by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. The VDA-DMA diblock copolymer was reacted with 2-(methylthio)ethylamine (MTEA) and 3-(methylthio)propylamine (MTPA) to yield two novel thioether functional diblock copolymers whose structure was confirmed using ¹H NMR and FTIR spectroscopy. Both diblock copolymers formed micelles (20–30 nm) in aqueous media as confirmed by dynamic light scattering (DLS) and transmission electron microscopy. The self-assembled micelles were loaded with Nile Red, a model hydrophobic drug to study their ROS-triggered release mechanism. On addition of hydrogen peroxide (H₂O₂), the most common ROS species, the hydrophobic thioether core of these micelles oxidized, and both diblock copolymers became more hydrophilic. This triggered their disassembly and subsequent cargo release as characterized by UV–visible spectroscopy. The Nile Red loaded micelles demonstrated similar in-vitro ROS-mediated release when exposed to endogenous oxidants in a model inflammation environment simulated by the presence of activated macrophages. The responsive nanomaterials developed in this article have promising potential as drug carriers in applications where ROS-triggered delivery of cargo is required such as in inflammatory conditions.     

Gaussian extended cubature formulae for polyharmonic functions

The purpose of this paper is to show certain links between univariate interpolation by algebraic polynomials and the representation of polyharmonic functions. This allows us to construct cubature formulae for multivariate functions having highest order of precision with respect to the class of polyharmonic functions. We obtain a Gauss type cubature formula that uses values of linear functionals (integrals over hyperspheres) and is exact for all -harmonic functions, and consequently, for all algebraic polynomials of variables of degree .

     

Higher Order Turán Inequalities

The celebrated Turán inequalities , where denotes the Legendre polynomial of degree , are extended to inequalities for sums of products of four classical orthogonal polynomials. The proof is based on an extension of the inequalities , which hold for the Maclaurin coefficients of the real entire function in the Laguerre-Pólya class, .