Negative Effects of Copper Oxide Nanoparticles on Carbon and Nitrogen Cycle Microbial Activities in Contrasting Agricultural Soils and in Presence of Plants

Metal-oxide nanoparticles (NPs) such as copper oxide (CuO) NPs offer promising perspectives for the development of novel agro-chemical formulations of pesticides and fertilizers. However, their potential impact on agro-ecosystem functioning still remains to be investigated. Here, we assessed the impact of CuO-NPs (0.1, 1, and 100 mg/kg dry soil) on soil microbial activities involved in the carbon and nitrogen cycles in five contrasting agricultural soils in a microcosm experiment over 90 days. Additionally, in a pot experiment, we evaluated the influence of plant presence on the toxicity of CuO-NPs on soil microbial activities. CuO-NPs caused significant reductions of the three microbial activities measured (denitrification, nitrification, and soil respiration) at 100 mg/kg dry soil, but the low concentrations (0.1 and 1 mg/kg) had limited effects. We observed that denitrification was the most sensitive microbial activity to CuO-NPs in most soil types, while soil respiration and nitrification were mainly impacted in coarse soils with low organic matter content. Additionally, large decreases in heterotrophic microbial activities were observed in soils planted with wheat, even at 1 mg/kg for soil substrate-induced respiration, indicating that plant presence did not mitigate or compensate CuO-NP toxicity for microorganisms. These two experiments show that CuO-NPs can have detrimental effects on microbial activities in soils with contrasting physicochemical properties and previously exposed to various agricultural practices. Moreover, we observed that the negative effects of CuO-NPs increased over time, indicating that short-term studies (hours, days) may underestimate the risks posed by these contaminants in soils

Functionally pathogenic EARS2 variants in vitro may not manifest a phenotype in vivo

OBJECTIVE: To investigate the genetic etiology of a patient diagnosed with leukoencephalopathy, brain calcifications, and cysts (LCC). METHODS: Whole-exome sequencing was performed on a patient with LCC and his unaffected family members. The variants were subject to in silico and in vitro functional testing to determine pathogenicity. RESULTS: Whole-exome sequencing uncovered compound heterozygous mutations in EARS2, c.328G>A (p.G110S), and c.1045G>A (p.E349K). This gene has previously been implicated in the autosomal recessive leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The p.G110S mutation has been found in multiple patients with LTBL. In silico analysis supported pathogenicity in the second variant. In vitro functional testing showed a significant mitochondrial dysfunction demonstrated by an ∼11% decrease in the oxygen consumption rate and ∼43% decrease in the maximum respiratory rate in the patient's skin fibroblasts compared with the control. EARS2 protein levels were reduced to 30% of normal controls in the patient's fibroblasts. These deficiencies were corrected by the expression of the wild-type EARS2 protein. However, a further unrelated genetic investigation of our patient revealed the presence of biallelic variants in a small nucleolar RNA (SNORD118) responsible for LCC. CONCLUSIONS: Here, we report seemingly pathogenic EARS2 mutations in a single patient with LCC with no biochemical or neuroimaging presentations of LTBL. This patient illustrates that variants with demonstrated impact on protein function should not necessarily be considered clinically relevant.Funding provided by the Baylor Scott & White Healthcare Foundation

Nitrous oxide emissions from European agriculture - An analysis of variability and drivers of emissions from field experiments

Nitrous oxide emissions from a network of agricultural experiments in Europe were used to explore the relative importance of site and management controls of emissions. At each site, a selection of management interventions were compared within replicated experimental designs in plot-based experiments. Arable experiments were conducted at Beano in Italy, El Encin in Spain, Foulum in Denmark, Logården in Sweden, Maulde in Belgium, Paulinenaue in Germany, and Tulloch in the UK. Grassland experiments were conducted at Crichton, Nafferton and Peaknaze in the UK, Gödöllö in Hungary, Rzecin in Poland, Zarnekow in Germany and Theix in France. Nitrous oxide emissions were measured at each site over a period of at least two years using static chambers. Emissions varied widely between sites and as a result of manipulation treatments. Average site emissions (throughout the study period) varied between 0.04 and 21.21 kg N₂O-N ha⁻¹ yr⁻¹, with the largest fluxes and variability associated with the grassland sites. Total nitrogen addition was found to be the single most important determinant of emissions, accounting for 15% of the variance (using linear regression) in the data from the arable sites (<i>p</i> < 0.0001), and 77% in the grassland sites. The annual emissions from arable sites were significantly greater than those that would be predicted by IPCC default emission factors. Variability of N₂O emissions within sites that occurred as a result of manipulation treatments was greater than that resulting from site-to-site and year-to-year variation, highlighting the importance of management interventions in contributing to greenhouse gas mitigation

Active site maturation and activity of the copper-radical oxidase GlxA is governed by a tryptophan residue

GlxA from Streptomyces lividans is a mononuclear copper-radical oxidase and a member of the auxiliary activity family 5 (AA5). Its domain organisation and low sequence homology make it a distinct member of the AA5 family in which the fungal galactose 6-oxidase (Gox) is the best-characterized. GlxA is a key cuproenzyme in the copper-dependent morphological development of S. lividans with a function that is linked to the processing of an extracytoplasmic glycan. The catalytic site in GlxA and Gox contain two distinct one-electron acceptors comprising the copper ion and a 3'-(S-cysteinyl) tyrosine. The latter is formed post-translationally through a covalent bond between a cysteine and a copper coordinating tyrosine ligand and houses a radical. In GlxA and Gox a second coordination sphere tryptophan residue (Trp288 in GlxA) is present, but the orientation of the indole ring differs between the two enzymes creating a marked difference in the ?-? stacking interaction of the benzyl ring with the 3'-(S-cysteinyl) tyrosine. Differences in the spectroscopic and enzymatic activity have been reported between GlxA and Gox with the indole orientation suggested as a reason. Here we report a series of in vivo and in vitro studies using the W288F and W288A variants of GlxA to assess the role of Trp288 on the morphology, maturation, spectroscopic and enzymatic properties. Our findings point towards a salient role for Trp288 in the kinetics of copper loading and maturation of GlxA, with its presence essential for stabilising the metalloradical site required for coupling catalytic activity and morphological development

Fluorescent mannosides serve as acceptor substrates for glycosyltransferase and sugar-1-phosphate transferase activities in <i>Euglena gracilis</i> membranes

Synthetic hexynyl α-D-mannopyranoside and its α-1,6-linked disaccharide counterpart were fluorescently labelled through CuAAC click chemistry with 3-azido-7-hydroxycoumarin. The resulting triazolyl-coumarin adducts, which were amenable to analysis by TLC, HPLC and mass spectrometry, proved to be acceptor substrates for α-1,6-ManT activities in mycobacterial membranes, as well as α- and β-GalT activities in trypanosomal membranes, benchmarking the potential of the fluorescent acceptor approach against earlier radiochemical assays. Following on to explore the glycobiology of the benign protozoan alga Euglena gracilis, α-1,3- and α-1,2-ManT activities were detected in membrane preparations, along with GlcT, Glc-P-T and GlcNAc-P-T activities. These studies serve to demonstrate the potential of readily accessible fluorescent glycans as substrates for exploring carbohydrate active enzymes

DendroBlast: approximate phylogenetic trees in the absence of multiple sequence alignments

The rapidly growing availability of genome information has created considerable demand for both fast and accurate phylogenetic inference algorithms. We present a novel method called DendroBLAST for reconstructing phylogenetic dendrograms/trees from protein sequences using BLAST. This method differs from other methods by incorporating a simple model of sequence evolution to test the effect of introducing sequence changes on the reliability of the bipartitions in the inferred tree. Using realistic simulated sequence data we demonstrate that this method produces phylogenetic trees that are more accurate than other commonly-used distance based methods though not as accurate as maximum likelihood methods from good quality multiple sequence alignments. In addition to tests on simulated data, we use DendroBLAST to generate input trees for a supertree reconstruction of the phylogeny of the Archaea. This independent analysis produces an approximate phylogeny of the Archaea that has both high precision and recall when compared to previously published analysis of the same dataset using conventional methods. Taken together these results demonstrate that approximate phylogenetic trees can be produced in the absence of multiple sequence alignments, and we propose that these trees will provide a platform for improving and informing downstream bioinformatic analysis. A web implementation of the DendroBLAST method is freely available for use at http://www.dendroblast.com/

Diversity of Beetle Genes Encoding Novel Plant Cell Wall Degrading Enzymes

Plant cell walls are a heterogeneous mixture of polysaccharides and proteins that require a range of different enzymes to degrade them. Plant cell walls are also the primary source of cellulose, the most abundant and useful biopolymer on the planet. Plant cell wall degrading enzymes (PCWDEs) are therefore important in a wide range of biotechnological processes from the production of biofuels and food to waste processing. However, despite the fact that the last common ancestor of all deuterostomes was inferred to be able to digest, or even synthesize, cellulose using endogenous genes, all model insects whose complete genomes have been sequenced lack genes encoding such enzymes. To establish if the apparent “disappearance” of PCWDEs from insects is simply a sampling problem, we used 454 mediated pyrosequencing to scan the gut transcriptomes of beetles that feed on a variety of plant derived diets. By sequencing the transcriptome of five beetles, and surveying publicly available ESTs, we describe 167 new beetle PCWDEs belonging to eight different enzyme families. This survey proves that these enzymes are not only present in non-model insects but that the multigene families that encode them are apparently undergoing complex birth-death dynamics. This reinforces the observation that insects themselves, and not just their microbial symbionts, are a rich source of PCWDEs. Further it emphasises that the apparent absence of genes encoding PCWDEs from model organisms is indeed simply a sampling artefact. Given the huge diversity of beetles alive today, and the diversity of their lifestyles and diets, we predict that beetle guts will emerge as an important new source of enzymes for use in biotechnology

A Bacterial Glucanotransferase Can Replace the Complex Maltose Metabolism Required for Starch to Sucrose Conversion in Leaves at Night

Peer reviewedPublisher PD

Properties of an alkali-thermo stable xylanase from Geobacillus thermodenitrificans A333 and applicability in xylooligosaccharides generation

An extracellular thermo-alkali-stable and cellulase-free xylanase from Geobacillus thermodenitrificans A333 was purified to homogeneity by ion exchange and size exclusion chromatography. Its molecular mass was 44 kDa as estimated in native and denaturing conditions by gel filtration and SDS-PAGE analysis, respectively. The xylanase (GtXyn) exhibited maximum activity at 70 °C and pH 7.5. It was stable over broad ranges of temperature and pH retaining 88 % of activity at 60 °C and up to 97 % in the pH range 7.5–10.0 after 24 h. Moreover, the enzyme was active up to 3.0 M sodium chloride concentration, exhibiting at that value 70 % residual activity after 1 h. The presence of other metal ions did not affect the activity with the sole exceptions of K+ that showed a stimulating effect, and Fe2+, Co2+ and Hg2+, which inhibited the enzyme. The xylanase was activated by non-ionic surfactants and was stable in organic solvents remaining fully active over 24 h of incubation in 40 % ethanol at 25 °C. Furthermore, the enzyme was resistant to most of the neutral and alkaline proteases tested. The enzyme was active only on xylan, showing no marked preference towards xylans from different origins. The hydrolysis of beechwood xylan and agriculture-based biomass materials yielded xylooligosaccharides with a polymerization degree ranging from 2 to 6 units and xylobiose and xylotriose as main products. These properties indicate G. thermodenitrificans A333 xylanase as a promising candidate for several biotechnological applications, such as xylooligosaccharides preparation