Yeast-based automated high-throughput screens to identify anti-parasitic lead compounds

We have developed a robust, fully automated anti-parasitic drug-screening method that selects compounds specifically targeting parasite enzymes and not their host counterparts, thus allowing the early elimination of compounds with potential side effects. Our yeast system permits multiple parasite targets to be assayed in parallel owing to the strains’ expression of different fluorescent proteins. A strain expressing the human target is included in the multiplexed screen to exclude compounds that do not discriminate between host and parasite enzymes. This form of assay has the advantages of using known targets and not requiring the in vitro culture of parasites. We performed automated screens for inhibitors of parasite dihydrofolate reductases, N-myristoyltransferases and phosphoglycerate kinases, finding specific inhibitors of parasite targets. We found that our ‘hits’ have significant structural similarities to compounds with in vitro anti-parasitic activity, validating our screens and suggesting targets for hits identified in parasite-based assays. Finally, we demonstrate a 60 per cent success rate for our hit compounds in killing or severely inhibiting the growth of Trypanosoma brucei, the causative agent of African sleeping sickness.     

Development of DNA methylation-based epigenetic age predictors in loblolly pine (Pinus taeda)

Biological ageing is connected to life history variation across ecological scales and informs a basic understanding of age-related declines in organismal function. Altered DNA methylation dynamics are a conserved aspect of biological ageing and have recently been modelled to predict chronological age among vertebrate species. In addition to their utility in estimating individual age, differences between chronological and predicted ages arise due to acceleration or deceleration of epigenetic ageing, and these discrepancies are linked to disease risk and multiple life history traits. Although evidence suggests that patterns of DNA methylation can describe ageing in plants, predictions with epigenetic clocks have yet to be performed. Here, we resolve the DNA methylome across CpG, CHG, and CHH-methylation contexts in the loblolly pine tree (Pinus taeda) and construct epigenetic clocks capable of predicting ages in this species within 6% of its maximum lifespan. Although patterns of CHH-methylation showed little association with age, both CpG and CHG-methylation contexts were strongly associated with ageing, largely becoming hypomethylated with age. Among age-associated loci were those in close proximity to malate dehydrogenase, NADH dehydrogenase, and 18S and 26S ribosomal RNA genes. This study reports one of the first epigenetic clocks in plants and demonstrates the universality of age-associated DNA methylation dynamics which can inform conservation and management practices, as well as our ecological and evolutionary understanding of biological ageing in plants.     

Polymerization on the Rocks: Negatively-Charged α-amino acids

Oligomers of the negatively-charged amino acids, glutamic acid, aspartic acid, and O-phospho-L-serine are adsorbed by hydroxylapatite and illite with affinities that increase with oligomer length. In the case of oligo-glutamic acids adsorbed on hydroxylapatite, addition of an extra residue results in an approximately four-fold increase in the strength of adsorption. Oligomers much longer than the 7-mer are retained tenaciously by the mineral. Repeated incubation of short oligo-glutamic acids adsorbed on hydroxylapatite or illite with activated monomer leads to the accumulation of oligomers at least 45 units long. The corresponding reactions of aspartic acid and O-phospho-L-serine on hydroxylapatite are less effective in generating long oligomers, while illite fails to accumulate substantial amounts of long oligomers of aspartic acid or of O-phospho-L-serine.     

Gene expression induced by copper stress in the diatom Thalassiosira pseudonana

Utilizing a PCR-based subtractive cDNA approach, we demonstrated that the marine diatom Thalassiosira pseudonana exhibits a rapid response at the gene level to elevated concentrations of copper and that this response attenuates over 24 h of continuous exposure. A total of 16 copper-induced genes were identified, 11 of which were completely novel; however, many of the predicted amino acid sequences had characteristics suggestive of roles in ameliorating copper toxicity. Most of the novel genes were not equivalently induced by H2O2- or Cd-induced stress, indicating specificity in response. Two genes that could be assigned functions based on homology were also induced under conditions of general cellular stress. Half of the identified genes were located within two inverted repeats in the genome, and novel genes in one inverted repeat had mRNA levels induced by approximately 500- to 2,000-fold by exposure to copper for 1 h. Additionally, some of the inverted repeat genes demonstrated a dose-dependent response to Cu, but not Cd, and appear to belong to a multigene family. This multigene family may be the diatom functional homolog of metallothioneins.     

Biodiversity improves the ecological design of sustainable biofuel systems

For algal biofuels to become a commercially viable and sustainable means of decreasing greenhouse gas emissions, growers are going to need to design feedstocks that achieve at least three characteristics simultaneously as follows: attain high yields; produce high quality biomass; and remain stable through time. These three qualities have proven difficult to achieve simultaneously under the ideal conditions of the laboratory, much less under field conditions (e.g., outdoor culture ponds) where feedstocks are exposed to highly variable conditions and the crop is vulnerable to invasive species, disease, and grazers. Here, we show that principles from ecology can be used to improve the design of feedstocks and to optimize their potential for “multifunctionality.” We performed a replicated experiment to test these predictions under outdoor conditions. Using 80 ponds of 1,100 L each, we tested the hypotheses that polycultures would outperform monocultures in terms of the following functions: biomass production, yield of biocrude from biomass, temporal stability, resisting population crashes, and resisting invasions by unwanted species. Overall, species richness improved stability, biocrude yield, and resistance to invasion. While this suggests that polycultures could outperform monocultures on average, invasion resistance was the only function where polycultures outperformed the best single species in the experiment. Due to tradeoffs among different functions that we measured, no species or polyculture was able to maximize all functions simultaneously. However, diversity did enhance the potential for multifunctionality—the most diverse polyculture performed more functions at higher levels than could any of the monocultures. These results are a key finding for ecological design of sustainable biofuel systems because they show that while a monoculture may be the optimal choice for maximizing short‐term biomass production, polycultures can offer a more stable crop of the desired species over longer periods of time.     

Production of high levels of 8:0 and 10:0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana

The Mexican shrub Cuphea hookeriana accumulates up to 75% caprylate (8:0) and caprate (10:0) in its seed oil. An acyl-ACP thioesterase cDNA from C. hookeriana , designated Ch FatB2 , has been identified, which, when expressed in Escherichia coli , provides thioesterase activity specific for 8:0- and 10:0-ACP substrates. Expression of this clone in seeds of transgenic canola, an oilseed crop that normally does not accumulate any 8:0 and 10:0, resulted in a dramatic increase in the levels of these two fatty acids accompanied by a preferential decrease in the levels of linoleate (18:2) and linolenate (18:3). The Ch FatB2 differs from Ch FatB1 , another Cuphea hookeriana thioesterase reported recently, in both substrate specificity and expression pattern. The Ch FatB1 has a broad substrate specificity with strong preference for 16:0-ACP and is expressed throughout the plant; whereas Ch FatB2 is specific for 8:0/10:0-ACP and its expression is confined to the seed. It is proposed that the amplified expression of Ch FatB2 in the embryo provides the hydrolytic enzyme specificity determining the fatty acyl composition of Cuphea hookeriana seed oil.