Parallel selection of antibody libraries on phage and yeast surfaces via a cross-species display

We created a cross-species display system that allows the display of the same antibody libraries on both prokaryotic phage and eukaryotic yeast without the need for molecular cloning. Using this cross-display system, a large, diverse library can be constructed once and subsequently used for display and selection in both phage and yeast systems. In this article, we performed the parallel phage and yeast selection of an antibody maturation library using this cross-display platform. This parallel selection allowed us to isolate more unique hits than single-species selection, with 162 unique clones from phage and 107 unique clones from yeast. In addition, we were able to shuttle yeast hits back to Escherichia coli cells for affinity characterization at a higher throughput.     

Design and synthesis of conformationally constrained cyclophilin inhibitors showing a cyclosporin-A phenotype in C. elegans

Cyclophilin A (CypA) is a member of the immunophilin family of proteins and receptor for the immunosuppressant drug cyclosporin A (CsA). Here we describe the design and synthesis of a new class of small-molecule inhibitors for CypA that are based upon a dimedone template. Electrospray mass spectrometry is utilised as an initial screen to quantify the protein affinity of the ligands. Active inhibitors and fluorescently labelled derivatives are then used as chemical probes for investigating the biological role of cyclophilins in the nematode Caenorhabditis elegans.     

Identification and Ruminal Outflow of Long-Chain Fatty Acid Biohydrogenation Intermediates in Cows Fed Diets Containing Fish Oil

The abundance of 20- to 24-carbon fatty acids in omasal digesta of cows fed grass silage-based diets supplemented with 0 (Control) and 250 g/day of fish oil (FO) was examined to investigate the fate of long-chain unsaturated fatty acids in the rumen. Complimentary argentation thin-layer chromatography and gas-chromatography mass-spectrometry analysis of fatty acid methyl esters and corresponding 4,4-dimethyloxazoline derivatives prepared from fish oil and omasal digesta enabled the structure of novel 20- to 22-carbon fatty acids to be elucidated. Compared with the Control, the FO treatment resulted in the formation and accumulation of 27 novel 20- and 22-carbon biohydrogenation intermediates containing at least one trans double bond and the appearance of cis-14 20:1, 20:2n-3, 21:4n-3 and 22:3n-6 not contained in fish oil. No conjugated ≥20-carbon fatty acids were detected in Control or FO digesta. In conclusion, fish oil in the diet results in the formation of numerous long-chain biohydrogenation intermediates in the rumen of lactating cows. Comparison of the intake and flow of 20-, 21- and 22-carbon fatty acids at the omasum in cows fed the Control and FO treatments suggests that the first committed steps of 20:5n-3, 21:5n-3 and 22:6n-3 hydrogenation in the rumen involve the reduction and/or isomerisation of double bonds closest to the carboxyl group.     

Crystallization of Fats: Influence of Minor Components and Additives

Over the years, there has been a steady stream of publications on the influence that minor components and additives have on the physical properties of fat continuous systems. These have been reviewed here. Both indigenous and added components are taken into account. The various materials have been discussed, ranging from partial glycerides and phospholipids to esterified sugars and polyols. Within the publications in this area, the (sub-)micron effects that these minor components have on nucleation, crystal growth, morphology, heat capacity and polymorphic stability have been described and discussed and, sometimes, explained. Similarly, the effects on a macroscopic level, such as visual aspects, melting profiles, post-hardening and rheology have been the subject of research. Although limited compositional information, especially of additives, hinders appropriate discussions of the relevant mechanisms, some generic guidelines as to what type and strength of effect can be expected have been derived. As a general rule, a more significant influence is observed when the acyl group of the minor component (where present) is similar to those present in the fat itself. Additives may have different effects depending on the fat they are added to, their concentration and the temperature, especially with increasing undercooling (which typically reduces the effect of additives).     

Effect of chain rigidity on block copolymer micelle formation and dissolution as observed by 1H-NMR spectroscopy

Amphiphilic copolymers poly(methyl methacrylate-b-acrylic acid), poly(methyl methacrylate-b-methacrylic acid), poly(methyl acrylate-b-acrylic acid) and poly(methyl acrylate-b-methacrylic acid) were prepared by reversible addition fragmentation chain-transfer (RAFT) polymerization. The hydrophilic polyacid blocks were either synthesized directly or formed by the hydrolysis of poly(tert-butyl acrylate) or poly(tert-butyl methacrylate) blocks. The hydrophobic blocks consisted of either the more rigid, high glass transition temperature (T _g ) poly(methyl methacrylate) or more flexible, low T _g poly(methyl acrylate) material. The hydrophilic blocks were either poly(methacrylic acid) (rigid, high T _g ) or poly(acrylic acid) (flexible, low T _g ). The micellization behavior of the polymers was studied by proton nuclear magnetic resonance (¹H-NMR) spectroscopy in mixtures of 1,4-dioxane-d₈ and D₂O. All four polymers were soluble in neat dioxane. In solutions of higher water content, the polymers with the more rigid hydrophobic blocks formed into micelles as was evidenced by broadening of the resonances resulting from the protons in those blocks. At moderate water concentration (25–50%), dissolution of the micelles was observed upon heating the solution. No micellization was observed in polymers containing the less rigid poly(methyl acrylate) hydrophobic block regardless of the identity of the hydrophilic block. As further evidence of micellization formation and dissolution, the spin-lattice (T ₁) and spin-spin (T ₂) relaxation times of protons in the hydrophobic and hydrophilic blocks were measured. Significant differences in the relaxation times as functions of temperature and solvent concentration were observed between the hydrophilic and hydrophobic blocks of the micelle-forming polymers.     

Precursor gas chemistry determines the crystallinity of carbon nanotubes synthesized at low temperature

Despite significant progress in carbon nanotube (CNT) synthesis by thermal chemical vapor deposition (CVD), the factors determining the structure of the resulting carbon filaments and other graphitic nanocarbons are not well understood. Here, we demonstrate that gas chemistry influences the crystal structure of carbon filaments grown at low temperatures (500 °C). Using thermal CVD, we decoupled the thermal treatment of the gaseous precursors (C₂H₄/H₂/Ar) and the substrate-supported catalyst. Varying the preheating temperature of the feedstock gas, we observed a striking transition between amorphous carbon nanofibers (CNFs) and crystalline CNTs. These results were confirmed using both a hot-wall CVD system and a cold-wall CVD reactor. Analysis of the exhaust gases (by ex situ gas chromatography) showed increasing concentrations of specific volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs) that correlated with the structural transition observed (characterized using high-resolution transmission electron microscopy). This suggests that the crystallinity of carbon filaments may be controlled by the presence of specific gas phase precursor molecules (e.g., VOCs and PAHs). Thus, direct delivery of these molecules in the CVD process may enable selective CNF or CNT formation at low substrate temperatures. The inherent scalability of this approach could impact many promising applications, especially in the electronics industry.