High land-use intensity exacerbates shifts in grassland vegetation composition after severe experimental drought.

Climate change projections anticipate increased frequency and intensity of drought stress, but grassland responses to severe droughts and their potential to recover are poorly understood. In many grasslands, high land-use intensity has enhanced productivity and promoted resource-acquisitive species at the expense of resource-conservative ones. Such changes in plant functional composition could affect the resistance to drought and the recovery after drought of grassland ecosystems with consequences for feed productivity resilience and environmental stewardship. In a 12-site precipitation exclusion experiment in upland grassland ecosystems across Switzerland, we imposed severe edaphic drought in plots under rainout shelters and compared them with plots under ambient conditions. We used soil water potentials to scale drought stress across sites. Impacts of precipitation exclusion and drought legacy effects were examined along a gradient of land-use intensity to determine how grasslands resisted to, and recovered after drought. In the year of precipitation exclusion, aboveground net primary productivity (ANPP) in plots under rainout shelters was -15% to -56% lower than in control plots. Drought effects on ANPP increased with drought severity, specified as duration of topsoil water potential ψ < -100 kPa, irrespective of land-use intensity. In the year after drought, ANPP had completely recovered, but total species diversity had declined by -10%. Perennial species showed elevated mortality, but species richness of annuals showed a small increase due to enhanced recruitment. In general, the more resource-acquisitive grasses increased at the expense of the deeper-rooted forbs after drought, suggesting that community reorganization was driven by competition rather than plant mortality. The negative effects of precipitation exclusion on forbs increased with land-use intensity. Our study suggests a synergistic impact of land-use intensification and climate change on grassland vegetation composition, and implies that biomass recovery after drought may occur at the expense of biodiversity maintenance.

Species richness effects on grassland recovery from drought depend on community productivity in a multisite experiment.

Biodiversity can buffer ecosystem functioning against extreme climatic events, but few experiments have explicitly tested this. Here, we present the first multisite biodiversity × drought manipulation experiment to examine drought resistance and recovery at five temperate and Mediterranean grassland sites. Aboveground biomass production declined by 30% due to experimental drought (standardised local extremity by rainfall exclusion for 72-98 consecutive days). Species richness did not affect resistance but promoted recovery. Recovery was only positively affected by species richness in low-productive communities, with most diverse communities even showing overcompensation. This positive diversity effect could be linked to asynchrony of species responses. Our results suggest that a more context-dependent view considering the nature of the climatic disturbance as well as the productivity of the studied system will help identify under which circumstances biodiversity promotes drought resistance or recovery. Stability of biomass production can generally be expected to decrease with biodiversity loss and climate change.

Artificial nucleotide codons for enzymatic DNA synthesis.

Herein, we report the high-yielding solid-phase synthesis of unmodified and chemically modified trinucleotide triphosphates (dN3TPs). These synthetic codons can be used for enzymatic DNA synthesis provided their scaffold is stabilized with phosphorothioate units. Enzymatic synthesis with three rather than one letter nucleotides will be useful to produce xenonucleic acids (XNAs) and for in vitro selection of modified functional nucleic acids.

Towards the controlled enzymatic synthesis of LNA containing oligonucleotides.

Enzymatic, de novo XNA synthesis represents an alternative method for the production of long oligonucleotides containing chemical modifications at distinct locations. While such an approach is currently developed for DNA, controlled enzymatic synthesis of XNA remains at a relative state of infancy. In order to protect the masking groups of 3'-O-modified LNA and DNA nucleotides against removal caused by phosphatase and esterase activities of polymerases, we report the synthesis and biochemical characterization of nucleotides equipped with ether and robust ester moieties. While the resulting ester-modified nucleotides appear to be poor substrates for polymerases, ether-blocked LNA and DNA nucleotides are readily incorporated into DNA. However, removal of the protecting groups and modest incorporation yields represent obstacles for LNA synthesis via this route. On the other hand, we have also shown that the template-independent RNA polymerase PUP represents a valid alternative to the TdT and we have also explored the possibility of using engineered DNA polymerases to increase substrate tolerance for such heavily modified nucleotide analogs.

Positive diversity-invasibility relationship in species-rich semi-natural grassland at the neighbourhood scale.

BACKGROUND AND AIMS: Attempts to answer the old question of whether high diversity causes high invasion resistance have resulted in an invasion paradox: while large-scale studies often find a positive relationship between diversity and invasibility, small-scale experimental studies often find a negative relationship. Many of the small-scale studies are conducted in artificial communities of even-aged plants. Species in natural communities, however, do not represent one simultaneous cohort and occur at various levels of spatial aggregation at different scales. This study used natural patterns of diversity to assess the relationship between diversity and invasibility within a uniformly managed, semi-natural community. METHODS: In species-rich grassland, one seed of each of ten species was added to each of 50 contiguous 16 cm(2) quadrats within seven plots (8 × 100 cm). The emergence of these species was recorded in seven control plots, and establishment success was measured in relation to the species diversity of the resident vegetation at two spatial scales, quadrat (64 cm(2)) within plots (800 cm(2)) and between plots within the site (approx. 400 m(2)) over 46 months. KEY RESULTS: Invader success was positively related to resident species diversity and richness over a range of 28-37 species per plot. This relationship emerged 7 months after seed addition and remained over time despite continuous mortality of invaders. CONCLUSIONS: Biotic resistance to plant invasion may play only a sub-ordinate role in species-rich, semi-natural grassland. As possible alternative explanations for the positive diversity-invasibility relationship are not clear, it is recommended that future studies elaborate fine-scale environmental heterogeneity in resource supplies or potential resource flows from resident species to seedlings by means of soil biological networks established by arbuscular mycorrhizal fungi.

Spatial heterogeneity confounded ozone-exposure experiment in semi-natural grassland.

Interpretation of observations from manipulative experiments is often complicated by a multitude of uncontrolled processes operating at various spatial and temporal scales. As such processes may differ among experimental plots there is a risk that effects of experimental treatments are confounded. Here we report on a free-air ozone-exposure experiment in permanent semi-natural grassland that suggested strong ozone effects on community productivity after 5 years. We tested ozone effects and investigated the potential of confounding due to changes in nutrient management. Repeated-measure ANOVA revealed mainly negative temporal trends for frequency of abundant productive plant species. Constrained ordination additionally showed converging trajectories of species compositions for ozone and control treatments with time. Yields sampled prior to the start of the experiment and soil nitrogen concentrations revealed that spatial heterogeneity in the soil nutrient status was not accounted for by the random allocation of treatments to plots with a bias towards less productive patches in the elevated-ozone plots. Re-analysis of yield data using repeated-measure ANOVA with a covariable to account for productivity prior to the start of fumigation revealed effects on the temporal changes in total yield and yield of legumes that cannot be separated between ozone and pre-treatment nutrient status. Changes in species composition favour an ecological interpretation with spatial heterogeneity as the major cause of different yield declines. Although elevated ozone may cause subtle physiological changes with longer term implications, our new results suggest that species-rich mature grassland such as the one studied at Le Mouret may be less sensitive to elevated ozone than previously assumed. In this experiment a confounded design was hidden at the start by transitory effects of a prior change in nutrient treatments.

Simultaneous determination of elbasvir and grazoprevir in fixed-dose combination and mass spectral characterization of each degradation product by UHPLC-ESI-QTOF-MS/MS.

Zepatier® (Elbasvir and Grazoprevir) is a novel two-drug, fixed-dose combination product containing elbasvir and grazoprevir used for the treatment of chronic hepatitis C virus infection. Various forced degradation studies of the two drugs had been conducted in order to identify significant degradation products and establish the degradation pathway induced by thermal, photolytic, acid/base hydrolytic and/or oxidative stress conditions. A reversed phase C18 UHPLC-PDA method has been developed for the analysis of the stressed samples. Seven significant degradation products of elbasvir and five significant degradation products of grazoprevir were found and investigated further by high resolution ESI-QTOF-MS with high accurate mass measurement (-1.96 to 1.36 ppm). The chemical structures of each degradation product were proposed based on their relative MS/MS fragmentation spectra in comparison with the corresponding parent drugs and the chemical synthetic knowledge of process chemists. The validated stability-indicating UHPLC method can be used in routine analysis for the simultaneous determination of elbasvir and grazoprevir in pharmaceutical formulations. As more and more combination drugs will enter into the market, this study can also shed light on stability indicating method development for combined drugs at early development stage.

Mean annual precipitation predicts primary production resistance and resilience to extreme drought.

Extreme drought is increasing in frequency and intensity in many regions globally, with uncertain consequences for the resistance and resilience of ecosystem functions, including primary production. Primary production resistance, the capacity to withstand change during extreme drought, and resilience, the degree to which production recovers, vary among and within ecosystem types, obscuring generalized patterns of ecological stability. Theory and many observations suggest forest production is more resistant but less resilient than grassland production to extreme drought; however, studies of production sensitivity to precipitation variability indicate that the processes controlling resistance and resilience may be influenced more by mean annual precipitation (MAP) than ecosystem type. Here, we conducted a global meta-analysis to investigate primary production resistance and resilience to extreme drought in 64 forests and grasslands across a broad MAP gradient. We found resistance to extreme drought was predicted by MAP; however, grasslands (positive) and forests (negative) exhibited opposing resilience relationships with MAP. Our findings indicate that common plant physiological mechanisms may determine grassland and forest resistance to extreme drought, whereas differences among plant residents in turnover time, plant architecture, and drought adaptive strategies likely underlie divergent resilience patterns. The low resistance and resilience of dry grasslands suggests that these ecosystems are the most vulnerable to extreme drought - a vulnerability that is expected to compound as extreme drought frequency increases in the future.

Metabolite identification via LC-SPE-NMR-MS of the in vitro biooxidation products of a lead mGlu5 allosteric antagonist and impact on the improvement of metabolic stability in the series.

Detailed information on the metabolic fate of lead compounds can be a powerful tool for an informed approach to the stabilization of metabolically labile compounds in the lead optimization phase. The combination of high performance liquid chromatography (HPLC) with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) has been used to give comprehensive structural data on metabolites of novel drugs in development. Recently, increased automation and the embedding of on-line solid-phase extraction (SPE) into a integrated LC-SPE-NMR-MS system have improved enormously the detection limits of this approach. The new technology platform allows the analysis of complex mixtures from microsome incubations, combining low material requirements with relatively high throughput. Such characteristics make it possible to thoroughly characterize metabolites of selected compounds at earlier phases along the path to lead identification and clinical candidate selection, thus providing outstanding guidance in the process of eliminating undesired metabolism and detecting active or potentially toxic metabolites. Such an approach was applied at the lead identification stage of a backup program on metabotropic glutamate receptor 5 (mGlu5) allosteric inhibition. The major metabolites of a lead 5-aminothiazole-4-carboxylic acid amide 1 were synthesized and screened, revealing significant in vitro activity and possible involvement in the overall pharmacodynamic behavior of 1. The information collected on the metabolism of the highly active compound 1 was pivotal to the synthesis of related compounds with improved microsomal stability.

Novel ketolide antibiotics with a fused five-membered lactone ring--synthesis, physicochemical and antimicrobial properties.

In an effort to find novel semisynthetic macrolides with extended antibacterial spectrum and improved activity we prepared a series of compounds based on commercially available clarithromycin, a potent and safe antimicrobial agent of outstanding clinical and commercial interest. According to the literature, improvement of antibacterial activity of erythromycin type antibiotics can be achieved by introduction of fused heterocycles such as cyclic carbonates or carbamates at positions 11 and 12 (such as in telithromycin). In the course of the work presented here, a similar, hitherto unprecedented set of compounds bearing a five-membered lactone ring fused to positions 11 and 12 was prepared based on carbon-carbon bond formation via intramolecular Michael addition of a [(hetero)arylalkylthio]acetic acid ester enolate to an alpha,beta-unsaturated ketone as the key step. Some of the ketolide compounds described in this paper were highly active against a representative set of erythromycin sensitive and erythromycin resistant test strains. The best compound showed a similar antimicrobial spectrum and comparable activity in vitro as well as in vivo as telithromycin. Furthermore, some physicochemical properties of these compounds were determined and are presented here. On the basis of these results, the novel ketolide lactones presented in this paper emerged as valuable lead compounds with comparable properties as the commercial ketolide antibacterial telithromycin (Ketek).