Interleukin‐4‐Clicked Surfaces Drive M2 Macrophage Polarization

Driving macrophage (M?) polarization into the M2 phenotype provides potential against inflammatory diseases. Interleukin‐4 (IL‐4) promotes polarization into the M2‐M? phenotype, but its systemic use is constrained by dose‐limiting toxicity. Consequently, we developed IL‐4‐decorated surfaces aiming at sustained and localized activity. IL‐4 muteins were generated by genetic code expansion; Lys42 was replaced by unnatural amino acids (uAAs). Both muteins showed cell‐stimulation ability and binding affinity to IL4Rα similar to those of wt‐IL‐4. Copper‐catalyzed (CuAAC) and copper‐free strain‐promoted (SPAAC) 1,3‐dipolar azide–alkyne cycloadditions were used to site‐selectively anchor IL‐4 to agarose surfaces. These surfaces had sustained IL‐4 activity, as demonstrated by TF‐1 cell proliferation and M2, but not M1, polarization of M‐CSF‐generated human M?. The approach provides a blueprint for the engineering of cytokine‐activated surfaces profiled for sustained and spatially controlled activity.     

Characterization of GPVI- or GPVI-CD39-Coated Nanoparticles and Their Impact on In Vitro Thrombus Formation

Traditional antithrombotic agents commonly share a therapy-limiting side effect, as they increase the overall systemic bleeding risk. A novel approach for targeted antithrombotic therapy is nanoparticles. In other therapeutic fields, nanoparticles have enabled site-specific delivery with low levels of toxicity and side effects. Here, we paired nanotechnology with an established dimeric glycoprotein VI-Fc (GPVI-Fc) and a GPVI-CD39 fusion protein, thereby combining site-specific delivery and new antithrombotic drugs. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles, NP-BSA, NP-GPVI and NP-GPVI-CD39 were characterized through electron microscopy, atomic force measurements and flow cytometry. Light transmission aggregometry enabled analysis of platelet aggregation. Thrombus formation was observed through flow chamber experiments. NP-GPVI and NP-GPVI-CD39 displayed a characteristic surface coating pattern. Fluorescence properties were identical amongst all samples. NP-GPVI and NP-GPVI-CD39 significantly impaired platelet aggregation. Thrombus formation was significantly impaired by NP-GPVI and was particularly impaired by NP-GPVI-CD39. The receptor-coated nanoparticles NP-GPVI and the bifunctional molecule NP-GPVI-CD39 demonstrated significant inhibition of in vitro thrombus formation. Consequently, the nanoparticle-mediated antithrombotic effect of GPVI-Fc, as well as GPVI-CD39, and an additive impact of CD39 was confirmed. In conclusion, NP-GPVI and NP-GPVI-CD39 may serve as a promising foundation for a novel therapeutic approach regarding targeted antithrombotic therapy.     

Filling the gap between Continuous and Discrete Time Dynamics of Autoregressive Processes

Discretization of continuous time autoregressive (AR) processes driven by a Brownian motion and embedding of discrete time AR sequences driven by a Gaussian white noise are classical issues. The article aims at establishing and using such discretization and embedding formulae between extended AR continuous time processes and discrete time sequences. The continuous-time processes are driven by either Brownian or jump processes, and may have random coefficients depending on time; Lévy-driven processes are also considered. The innovation of the discrete time processes may be of many types – including Gaussian. In one way, observing the continuous time AR process at discrete times leads the AR dynamics of the discretized process to be characterized. The other way round, AR sequences can be embedded, in the almost sure sense, into continuous time AR processes with the same dynamics. Illustration is provided through many examples and simulation.     

Horizontal Dip‐Spin Casting of aqueous alumina‐polyvinylpyrrolidone suspensions with chopped fiber

A novel ceramic processing method, called Horizontal Dip Spin Casting (HDSC), enabled fabrication of tubular ceramic parts with an aligned chopped fiber phase. HDSC was demonstrated using highly loaded aqueous alumina suspensions with >50 vol.% solids loading and ≤5 vol.% water‐soluble polymer employed as a rheological modifier. Chopped carbon fibers were added to the suspensions to attain maximum loadings of 30 vol.%. During forming, cylindrical foam molds were dipped into the suspension while being rotated radially about the long axis. Simultaneously, a doctor blade was placed at a specified distance from the foam surface to facilitate the flow of the suspension to align the fiber and control the thickness of the material that accrued on the mold. Rheological study of alumina‐PVP suspensions with and without chopped carbon fiber showed that the suspensions exhibited a yield‐pseudoplastic flow behavior. The degree of alignment of the carbon fiber phase in the green bodies was characterized for various suspension formulations, carbon fiber contents and forming speeds. Stereological characterization of green body specimens confirmed the effectiveness of HDSC to attain the desired tubular geometry with considerable fiber alignment for a suspension composition containing ≤20 vol.% chopped fibers.     

A graphical representation for a statistical test of average equivalence and variance comparison with process data

This paper provides a graphical approach for presenting results of a statistical test of average equivalence with process data. In addition, a graphical representation of process change in variance is presented. An example from the biopharmaceutical industry is provided to illustrate the information contained in the chart. Copyright © 2010 John Wiley & Sons, Ltd.     

Isotopic identification of the sources of lead contamination for white storks (Ciconia ciconia) in a marshland ecosystem (Doñana,S.W. Spain)

White storks (Ciconia ciconia) fed in contaminated waters resulting from the Aznacollar acid mining-sludge spillage into the R. Guadiamar, which feeds the eastern flank of the Guadalquivir marshes (Do?ana), S.W. Spain. The sludge was rich in a range of toxic elements, and in organic pollutants such as the aromatic amines. Storks did not exhibit elevated metals in their blood immediately following the accident, but chick blood collected the year following the accident showed genotoxic damage compared to the controls. In this study lead isotope analysis was used to assess if the storks had ingested sludge-derived contaminants. The sludge lead isotope ratio was distinct from that of the Do?ana sediments. The stork blood lead isotope ratios exactly matched that of the sludge. It was concluded that the storks had ingested sludge-derived contaminants. A detailed study of the lead contamination along the R. Guadiamar and the R. Guadalquivir (of which the Guadiamar is a tributary) was also conducted to place the white stork colony lead exposure in the context of the spatial contamination of the storks' habitat.     

Cation/H+ antiporters mediate potassium and sodium fluxes in Pichia sorbitophila. Cloning of the PsNHA1 and PsNHA2 genes and expression in Saccharomyces cerevisiae

Pichia sorbitophila grows rapidly in the presence of very high NaCl concentrations. Under these conditions, even when the K(+) concentration is low, P. sorbitophila cells can maintain low Na(+) and high K(+) contents. This remarkable capacity of P. sorbitophila fails when the external pH is not acidic. This indicates that Na(+) efflux is mediated by an electroneutral Na(+)/H(+) antiporter. We have cloned and sequenced two genes designated as PsNHA1 and PsNHA2, which probably encode two antiporters of this type. The genes present high similarity with the corresponding genes from other yeasts. The heterologous expression of PsNHA1 or PsNHA2 in a Saccharomyces cerevisiae mutant lacking the Na(+) efflux systems and sensitive to high concentrations of Na(+) and K(+) rescued the tolerance and the ability to extrude both cations. The Accession Nos of the sequenced DNA fragments are: PsNHA1, AJ496431; PsNHA2, AJ496432. (TC 2.A.36) Copyright 2002 John Wiley & Sons, Ltd.     

Simulations of biomolecules: Characterization of the early steps in the pH-induced conformational conversion of the hamster,bovine and human forms of the prion protein

As computer power increases, so too does the range of interesting biomolecular phenomena and properties that can be simulated. It is now possible to simulate complicated protein conformational changes at ambient or physiological temperatures. In this regard, we are attempting to map the conformational transitions of the normal, cellular prion protein (PrP(C)) to its infectious scrapie isoform (PrP(Sc)), which causes neurodegenerative diseases in many mammals. These two forms have identical sequences and are conformational isomers, with heightened formation of beta-sheet structure in the scrapie form. Conversion can be triggered by lowering the pH, but thus far it has been impossible to characterize the conformational change at high resolution using experimental methods. Therefore, to investigate the effect of acidic pH on PrP conformation, we have performed molecular-dynamics simulations of hamster, human and bovine forms of the prion protein in water at neutral and low pH. In all cases the core of the protein is well maintained at neutral pH. At low pH, however, the protein is more dynamic, and the sheet-like structure increases both by lengthening of the native beta-sheet and by addition of a portion of the N-terminus to widen the sheet by another 2-3 strands.     

Identification of cross-linked peptides for protein interaction studies using mass spectrometry and 18O labeling

A new method is presented to screen proteolytic mass maps of cross-linked protein complexes for the presence of cross-linked peptides and for the verification of proposed structures. On the basis of the incorporation of 18O from isotopically enriched water into the C-termini of proteolytic peptides, cross-linked peptides are readily distinguished in mass spectra by a characteristic 8 amu shift. This is due to the incorporation of two 18O atoms in each C-terminus, so that normal and surface-labeled peptides shift 4 amu and cross-linked peptides containing two C-termini will shift 8 amu compared with their unlabeled counterparts. The method is fast, sensitive, and reliable and can be combined with any available cross-linking reagent and a wide range of proteolytic agents. As proof of principle, we successfully applied the method to a complex of two DNA repair proteins (Rad18-Rad6) and identified the interaction domain.