An ultrasensitive, continuous assay for xylanase using the fluorogenic substrate 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside

We describe a fluorescence-based assay for the analysis of xylanase activity using a novel fluorogenic substrate, 6,8-difluoro-4-methylumbelliferyl beta-D-xylobioside (DiFMUX(2)). Generation of fluorescent 6,8-difluoro-4-methylumbelliferone (DiFMU) from the substrate corresponded directly to xylanase activity. High-performance liquid chromatography analysis of the digestion products showed that xylanase hydrolyzed DiFMUX(2) directly to DiFMU and xylobiose. The assay provides the speed, sensitivity, and convenience required for measuring xylanase activity or for screening xylanase inhibitors in a high-throughput format and is suitable for the kinetic assay of xylanases from a variety of sources.     

Capillary electrophoresis for studying drug-DNA interactions

The development of new drugs to treat disease by binding directly to DNA offers much promise but is reliant on methods to determine the relative affinity of the putative drug for different DNA sequences. Such methods should ideally be rapid and inexpensive as well as reliable. Use of capillary electrophoresis in simple silica columns offers such a method. The development of systems in which the solvent carries a soluble polymer allows the reliable separation of DNA oligomers, of 12-20 bp in length, which can then be titrated with the ligand in competition experiments. The results obtained are comparable with those obtained by footprinting and give direct graphical output, easily analysed for relative binding affinity.     

Engineering of the methylmalonyl-CoA metabolite node of Saccharopolyspora erythraea for increased erythromycin production

Engineering of the methylmalonyl-CoA (mmCoA) metabolite node of the Saccharopolyspora erythraea wild-type strain through duplication of the mmCoA mutase (MCM) operon led to a 50% increase in erythromycin production in a high-performance oil-based fermentation medium. The MCM operon was carried on a 6.8kb DNA fragment in a plasmid which was inserted by homologous recombination into the S. erythraea chromosome. The fragment contained one uncharacterized gene, ORF1; three MCM related genes, mutA, mutB, meaB; and one gntR-family regulatory gene, mutR. Additional strains were constructed containing partial duplications of the MCM operon, as well as a knockout of ORF1. None of these strains showed any significant alteration in their erythromycin production profile. The combined results showed that increased erythromycin production only occurred in a strain containing a duplication of the entire MCM operon including mutR and a predicted stem-loop structure overlapping the 3' terminus of the mutR coding sequence.     

Stem growth characteristics of high yielding Miscanthus correlate with yield,development and intraspecific competition within plots

High yielding perennial grasses are utilized as biomass for the bioeconomy and to displace fossil fuels. Many such grasses, including Miscanthus, are largely undomesticated. The main Miscanthus crop is a naturally occurring hydrid M. × giganteus (Mxg). All above ground biomass from Miscanthus is harvested. Stem traits correlate strongly with yield and therefore understanding the seasonal progression of stem growth should identify routes for improved yield. If such studies utilized high yielding commercial genotypes growing in plots the conclusions are likely to be more commercially relevant. Stem elongation was measured from five high yielding genotypes, 10 plants per plot from 20 plots in a replicated field trial over 4 years. Richards growth function produced an accurate fit to stem elongation. Differentials, double differentials and integrals of the parameterized function produced six growth characteristics, describing growth rate, timing and duration of the logarithmic growth phase and area under the growth curve. Maximum growth rate was correlated with yield and compensatory interactions were identified, for example plants with higher maximal growth rates had shorter durations of logarithmic growth. Plant position within plots of lower yielding genotypes did not affect growth characteristics but had a significant effect on late season growth characteristics in higher yielding genotypes. Two high yielding genotypes were compared over 3 years and growth parameterized using four different factors. The inverse correlation between maximum growth rate and duration of logarithmic growth was consistent across years and factors in both genotypes except when parameterized using temperature and only in Mxg. This suggested that different limitations to growth were exerted on the two genotypes which may help explain the exceptional performance of the Mxg genotype. We discuss the implications of the identified complex interactions in growth characteristics for approaches to maximize seasonal yield in perennial biomass crops.     

Correction to: Contrasting rhizosphere soil nutrient economy of plants associated with arbuscular mycorrhizal and ectomycorrhizal fungi in karst forests

Plant and Soil - A Correction to this paper has been published: https://doi.org/10.1007/s11104-021-05032-6     

Size at birth, fasting glucose and insulin levels and insulin resistance in adult twins.

Studies in singletons have found an association between birthweight and Type 2 diabetes in adult life. The aim of this study was to investigate whether this association could also be seen in twins. 59 monozygotic (MZ) and 69 dizygotic (DZ) same-sex twin pairs aged 19-50 years and 89 singleton controls matched for age, gestational age, gender, maternal age and parity were recruited from a local obstetric database. Associations between adult glucose, HbA(1)C and insulin levels and insulin resistance and birthweight were assessed by linear regression with adjustment for confounding variables. Twins were significantly lighter at birth than singleton controls, but there were no significant differences in adult weight, glucose, HbA(1)C and insulin levels or insulin resistance between twins and controls. The relationship between birthweight and fasting glucose and insulin levels, and insulin resistance was not significantly different from zero in either twins or controls, but birthweight was significantly negatively associated with HbA(1)C only in controls. There was no evidence of a difference between MZ and DZ twins in unpaired or within-pair analysis. These results provide little evidence that low birthweight in twins increases the risk of impaired glucose-insulin metabolism in young adults or that genetic factors can account for the association observed in singletons.     

Bayesian inference of species diffusion in the West African Agama agama species group (Reptilia,Agamidae)

The savannah and tropical forest biomes of Africa have a long history of expansion and contraction, and the recent and rapid spread of dry savannah habitats has influenced the spatial and temporal diversification of vertebrate taxa across this region. We used a combination of species tree and phylogeographic methods to describe the spatio-temporal changes through time and across space (= species diffusion) in a clade of seven West African lizard species in the Agama agama species group. A Bayesian species tree diffusion approach was used to compare the relative rates at which species ranges changed across the landscape. We found that some species have high diffusion rates characterized by significant movement in their range location and minor changes to their overall range size, whereas other species show little movement in their range centre with an exponential increase in range size. This discrepancy between the rates that range locations shift versus change in their relative area could be linked to populations tracking their preferred habitats through time. A continuous Bayesian phylogeography approach using a relaxed random walk model was used to estimate the timing and rate of population size change and geographic diffusion in A. picticauda, the single species in the group with an extensive African distribution from Mauritania to Ethiopia. The mean dispersal rate of A. picticauda increased dramatically throughout the Pleistocene, and a Bayesian skyride analysis supports exponential population growth over this same time period. A comparison of genetic diversity across different loci and species suggests that A. lebretoni experienced a mitochondrial selective sweep that has caused a deficit of variation at this locus in relation to nuclear loci.     

Finding their niche: chemokines directing cell migration in the thymus

T lymphocytes are generated throughout life, arising from bone marrow-derived progenitors that complete an essential developmental process in the thymus. Thymic T cell education leads to the generation of a self-restricted and largely self-tolerant peripheral T-cell pool and is facilitated by interactions with thymic stromal cells residing in distinct supportive niches. The signals governing thymocyte precursor migration into the thymus, directing thymocyte navigation through thymic microenvironments and mature T-cell egress into circulation were, until recently, largely unknown, but presumed to be mediated to a large extent by chemokine signalling. Recent studies have now uncovered various specific functions for members of the chemokine superfamily in the thymus. These studies have not only revealed distinct but also in some cases overlapping roles for several chemokine family members in various thymocyte migration events and have also shown that homing and positioning of other cells in the thymus, such as dendritic cells and natural killer T cells is also chemokine-dependent. Here, we discuss current understanding of the role of chemokines in the thymus and highlight key future avenues for investigation in this field.