Metabolic engineering of Saccharomyces cerevisiae for production of Eicosapentaenoic Acid, using a novel {Delta}5-Desaturase from Paramecium tetraurelia

Very-long-chain polyunsaturated fatty acids, such as arachidonic acid (ARA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), have well-documented importance in human health and nutrition. Sustainable production in robust host organisms that do not synthesize them naturally requires the coordinated expression of several heterologous desaturases and elongases. In the present study we show production of EPA in Saccharomyces cerevisiae using glucose as the sole carbon source through expression of five heterologous fatty acid desaturases and an elongase. Novel Δ5-desaturases from the ciliate protozoan Paramecium tetraurelia and from the microalgae Ostreococcus tauri and Ostreococcus lucimarinus were identified via a BLAST search, and their substrate preferences and desaturation efficiencies were assayed in a yeast strain producing the ω6 and ω3 fatty acid substrates for Δ5-desaturation. The Δ5-desaturase from P. tetraurelia was up-to-2-fold more efficient than the microalgal desaturases and was also more efficient than Δ5-desaturases from Mortierella alpina and Leishmania major. In vivo investigation of acyl carrier substrate specificities showed that the Δ5-desaturases from P. tetraurelia, O. lucimarinus, O. tauri, and M. alpina are promiscuous toward the acyl carrier substrate but prefer phospholipid-bound substrates. In contrast, the Δ5-desaturase from L. major showed no activity on phospholipid-bound substrate and thus appears to be an exclusively acyl coenzyme A-dependent desaturase.     

Quantifying Traces of Tool Use: A Novel Morphometric Analysis of Damage Patterns on Percussive Tools

Percussive technology continues to play an increasingly important role in understanding the evolution of tool use. Comparing the archaeological record with extractive foraging behaviors in nonhuman primates has focused on percussive implements as a key to investigating the origins of lithic technology. Despite this, archaeological approaches towards percussive tools have been obscured by a lack of standardized methodologies. Central to this issue have been the use of qualitative, non-diagnostic techniques to identify percussive tools from archaeological contexts. Here we describe a new morphometric method for distinguishing anthropogenically-generated damage patterns on percussive tools from naturally damaged river cobbles. We employ a geomatic approach through the use of three-dimensional scanning and geographical information systems software to statistically quantify the identification process in percussive technology research. This will strengthen current technological analyses of percussive tools in archaeological frameworks and open new avenues for translating behavioral inferences of early hominins from percussive damage patterns.     

Power through ‘Us’: Leaders’ Use of We-Referencing Language Predicts Election Victory

Leaders have been observed to use distinct rhetorical strategies, but it is unclear to what extent such strategies are effective. To address this issue we analyzed the official election campaign speeches of successful and unsuccessful Prime Ministerial candidates in all 43 Australian Federal elections since independence from Britain in 1901 and measured candidates'' use of personal (‘I’, ‘me’) and collective pronouns (‘we’, ‘us’). Victors used more collective pronouns than their unsuccessful opponents in 80% of all elections. Across all elections, victors made 61% more references to ‘we’ and ‘us’ and used these once every 79 words (vs. every 136 words for losers). Extending social identity theorizing, this research suggests that electoral endorsement is associated with leaders'' capacity to engage with, and speak on behalf of, a collective identity that is shared with followers whose support and energies they seek to mobilize.     

In vino veritas: Oligomeric procyanidins and the ageing of red wines

The role of oligomeric procyanidins in the ageing of wine has been examined by the addition of these substances to red and white wines. Observations show that in wines the acid-catalysed C  C bond-breaking and bond-making process, characteristic of procyanidin chemistry, is set up and leads to the eventual precipitation of polymers. In red wines, procyanidin polymers are also additionally precipitated by reaction possibly with anthocyanins or products derived therefrom.     

Plant polyphenols—secondary metabolism and chemical defence: Some observations

Secondary metabolism and theories of plant-animal co-evolution are briefly discussed. Current ideas concerning the role of polyphenols as mediators of a general form of chemical defence in plants are outlined. Studies are described of the association of a group of biosynthetically inter-related polyphenols based on gallic acid and D-glucose with the protein bovine serum albumin. The results are interpreted in terms of structure-activity relationships in protein-polyphenol complexation and are used to comment on the co-evolution theory and the position of plant polyphenols in higher plant secondary metabolism and defence.     

Homogenization of geographical variants at the nontranscribed spacer of rDNA in Drosophila mercatorum

rDNA nontranscribed spacer (NTS) lengths of Drosophila mercatorum have been measured in individuals from several geographic regions. Individuals from the different geographic subpopulations share some length fragments but are in general distinct. The length differences, both within and between individuals, arise from different copy numbers of a 250-bp repeating unit that is localized to one part of the NTS. In addition to the length differences caused by the 250-bp repeat, there is a Y chromosome (male)-specific length variant elsewhere in the NTS that is approximately 70 bp shorter than the NTS fragment from the X chromosome. Sexual dimorphism seems to be present in all Drosophila. Also, D. mercatorum has fewer NTS length variants per individual than does D. melanogaster while possessing comparable levels of restriction- site polymorphism. The mechanisms that may cause this pattern of variation are selection, gene conversion, and unequal recombination.      

Towards automation of glycomic profiling of complex biological materials

Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.     

The influence of optimization target selection on the structure of arterial tree models generated by constrained constructive optimization

The computational method of constrained constructive optimization was used to generate complex arterial model trees by optimization with respect to a target function. Changing the target function also changes the tree structure obtained. For a parameterized family of target functions a series of trees was created, showing visually striking differences in structure that can also be quantified by appropriately chosen numerical indexes. Blood transport path length, pressure profile, and an index for relative segment orientation show clear dependencies on the optimization target, and the nature of changes can be explained on theoretical grounds. The main goal was to display, quantify, and explain the structural changes induced by different optimization target functions.     

Molecular dissection of PapD interaction with PapG reveals two chaperone-binding sites

P pili are composite adhesive fibres that allow uropathogenic Escherichia coli to gain a foothold in the host by binding to receptors present on the uroepithalium via the adhesin PapG. The assembly of P pili requires a periplasmic chaperone, PapD, that has an immunoglobulin-like three-dimensional structure. PapD-subunit complex formation involves a conserved anchoring mechanism in the chaperone cleft and a‘molecular zippering’to the extreme C-terminus of pilus subunits. A chaperone-binding assay was developed using fusions of the C-terminus of PapG to maltose-binding protein (MBP/G fusions) to investigate whether chaperone-subunit complex formation requires additional interactions. PapD bound strongly to an MBP/G fusion containing the C-terminal 140 amino acids of PapG (MBP/G175-314) but only weakly to the MBP/G234-314 fusion containing 81 C-terminal residues, arguing that the region between residues 175-234 contains additional information that is required for strong PapD-PapG interactions. PapD was shown to interact with a PapG C-terminal truncate containing residues 1-198 but not a truncate containing residues 1-145, suggesting the presence of a second, independent PapD interactive site. Four peptides overlapping the second site region were tested for binding to PapD in vitro to further delineate this motif. Only one of the peptides synthesized was recognized by PapD. The MBP/G fusion containing both binding sites formed a tight complex with PapD in vivo and inhibited pilus assembly by preventing chaperone-subunit complex formation.