Enzymatic assays: optimization of systems by using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes

A method to optimize enzymatic assays by using pyruvate kinase and lactate dehydrogenase enzymes is presented and applied to mitochondrial ATPase as an example. Optimum amounts of auxiliary enzymes, to obtain either a 99% of the initial rate in a given time (t99) or a given lag period (L), are calculated from their apparent Michaelis constants (Kapp) in the medium used and their prices per enzymatic international unit.     

Does fragmentation of wetlands affect gene flow in sympatric Acrocephalus warblers with different migration strategies?

Wetlands are naturally patchy habitats, but patchiness has been accentuated by the extensive wetlands loss due to human activities. In such a fragmented habitat, dispersal ability is especially important to maintain gene flow between populations. Here we studied population structure, genetic diversity and demographic history of Iberian and North African populations of two wetland passerines, the Eurasian reed warbler Acrocephalus scirpaceus and the moustached warbler Acrocephalus melanopogon. These species are closely related and sympatric in our study sites, but the reed warbler is a widespread long‐distance migrant while the moustached warbler's breeding range is patchier and it is resident or migrates over short distances. Using microsatellite and mtDNA data, we found higher differentiation in moustached than in reed warblers, indicating higher dispersal capability of the latter species. Our results also suggest that the sea limits dispersal in the moustached warbler. However, we found evidence of gene flow between the study sites in both species, indicating a capability to compensate for habitat fragmentation. In most cases, the gene flow was restricted, possibly because of the large distances between study sites (from ca 290 to 960 km) or breeding site fidelity. The reed warbler had higher haplotype diversity, likely due to dispersal from different populations, past admixture event and a larger population size. We found also signs of postglacial population growth for both species and evidence of a recent colonization or re‐colonization of the Mallorca Island by the moustached warbler.     

Impact of dispersal status on estimates of local population growth rates in a Temminck's stint Calidris temminckii population

Due to different costs and benefits associated with dispersal and philopatry, life history traits of immigrants and philopatric individuals may differ. Despite of the apparent effects, dispersal status is only rarely considered in analyses of population dynamics. We analysed whether dispersal status explains variation in life history traits of an endangered Temminck's stint Calidris temminckii population breeding at the Baltic Sea. We also estimated the impact of immigration and dispersal status on the population growth rate (λ) with a population matrix model, in which immigrants and philopatric individuals are separated to their own stages. We found that philopatric individuals had a higher apparent survival than immigrants in both sexes. In reproductive parameters, variation due to dispersal status was not clear. Nests incubated by philopatric individuals survived better than those of immigrants, but this did not translate in hatchling production per breeding attempt. Models described a sink population in which the inclusion of both immigration rate to the population and the dispersal status of individuals into the model increased estimates of λ. When the better success of philopatric individuals was considered, the population growth appeared more stable (λ=0.972). If this was not taken into account, λ implied a strong decline (λ=0.911). The results support the hypothesis that immigrants exhibit lower components of lifetime reproductive success and therefore contribute less to population growth and the gene pool than local recruits. While we cannot distinguish whether this difference reflects higher mortality or permanent emigration, the latter explanation seems more plausible. Our results highlight the importance of considering immigration and dispersal status in population modelling. In the case of the endangered study population, the results implied that management options directed to improve local recruitment would be a profitable option.     

Preplant release of Nesidiocoris tenuis and supplementary tactics for control of Tuta absoluta and Bemisa tabaci in greenhouse tomato

An early release system developed for Nesidiocoris tenuis Reuter (Heteroptera: Miridae) could provide a good control of Tuta absoluta (Meyrick) (Lepidoptera: Gelechiidae) in tomato. Tuta absoluta and the whitefly Bemisia tabaci Gennadius (Hemiptera: Aleyrodidae) often appear simultaneously in tomato crops and this might affect control capacity. Therefore, the new approach needs to be tested in a situation with both pests present. In addition, Bacillus thuringiensis Berliner and Trichogramma achaeae Nagaraja & Nagarkatti (Hymenoptera: Trichogrammatidae) have been shown to be effective against T. absoluta and could be a supplement to N. tenuis. Two experiments were carried out to evaluate the potential of this approach and its combination with supplementary control agents against T. absoluta. In the first experiment four treatments were compared (T. absoluta, B. tabaci, T. absoluta + N. tenuis, and T. absoluta + B. tabaci + N. tenuis) and N. tenuis was able to control T. absoluta and B. tabaci either alone or together. In the second experiment, five treatments were compared: T. absoluta, T. absoluta + N. tenuis, T. absoluta + N. tenuis + T. achaeae, T. absoluta + N. tenuis + B. thuringiensis, and T. absoluta + N. tenuis + T. achaeae + B. thuringiensis. Nesidiocoris tenuis again proved capable of significantly reducing T. absoluta populations, and the implementation of additional agents did not increase its effectiveness.     

Computer-Aided Design of Fragment Mixtures for NMR-Based Screening

Fragment-based drug discovery is widely applied both in industrial and in academic screening programs. Several screening techniques rely on NMR to detect binding of a fragment to a target. NMR-based methods are among the most sensitive techniques and have the further advantage of yielding a low rate of false positives and negatives. However, NMR is intrinsically slower than other screening techniques; thus, to increase throughput in NMR-based screening, researchers often assay mixtures of fragments, rather than single fragments. Herein we present a fast and straightforward computer-aided method to design mixtures of fragments taken from a library that have minimized NMR signal overlap. This approach enables direct identification of one or several active fragments without the need for deconvolution. Our approach entails encoding of NMR spectra into a computer-readable format that we call a fingerprint, and minimizing the global signal overlap through a Monte Carlo algorithm. The scoring function used favors a homogenous distribution of the global signal overlap. The method does not require additional experimental work: the only data required are NMR spectra, which are generally recorded for each compound as a quality control measure before its insertion into the library.     

Microbial diversity in the midguts of field and lab-reared populations of the European corn borer Ostrinia nubilalis

BACKGROUND: Insects are associated with microorganisms that contribute to the digestion and processing of nutrients. The European Corn Borer (ECB) is a moth present world-wide, causing severe economical damage as a pest on corn and other crops. In the present work, we give a detailed view of the complexity of the microorganisms forming the ECB midgut microbiota with the objective of comparing the biodiversity of the midgut-associated microbiota and explore their potential as a source of genes and enzymes with biotechnological applications. METHODOLOGICAL/PRINCIPAL FINDINGS: A high-throughput sequencing approach has been used to identify bacterial species, genes and metabolic pathways, particularly those involved in plant-matter degradation, in two different ECB populations (field-collected vs. lab-reared population with artificial diet). Analysis of the resulting sequences revealed the massive presence of Staphylococcus warneri and Weissella paramesenteroides in the lab-reared sample. This enabled us to reconstruct both genomes almost completely. Despite the apparently low diversity, 208 different genera were detected in the sample, although most of them at very low frequency. By contrast, the natural population exhibited an even higher taxonomic diversity along with a wider array of cellulolytic enzyme families. However, in spite of the differences in relative abundance of major taxonomic groups, not only did both metagenomes share a similar functional profile but also a similar distribution of non-redundant genes in different functional categories. CONCLUSIONS/SIGNIFICANCE: Our results reveal a highly diverse pool of bacterial species in both O. nubilalis populations, with major differences: The lab-reared sample is rich in gram-positive species (two of which have almost fully sequenced genomes) while the field sample harbors mainly gram-negative species and has a larger set of cellulolytic enzymes. We have found a clear relationship between the diet and the midgut microbiota, which reveals the selection pressure of food on the community of intestinal bacteria.     

Metabolic networks of Sodalis glossinidius: a systems biology approach to reductive evolution

Background

Genome reduction is a common evolutionary process affecting bacterial lineages that establish symbiotic or pathogenic associations with eukaryotic hosts. Such associations yield highly reduced genomes with greatly streamlined metabolic abilities shaped by the type of ecological association with the host. Sodalis glossinidius, the secondary endosymbiont of tsetse flies, represents one of the few complete genomes available of a bacterium at the initial stages of this process. In the present study, genome reduction is studied from a systems biology perspective through the reconstruction and functional analysis of genome-scale metabolic networks of S. glossinidius.

Results

The functional profile of ancestral and extant metabolic networks sheds light on the evolutionary events underlying transition to a host-dependent lifestyle. Meanwhile, reductive evolution simulations on the extant metabolic network can predict possible future evolution of S. glossinidius in the context of genome reduction. Finally, knockout simulations in different metabolic systems reveal a gradual decrease in network robustness to different mutational events for bacterial endosymbionts at different stages of the symbiotic association.

Conclusions

Stoichiometric analysis reveals few gene inactivation events whose effects on the functionality of S. glossinidius metabolic systems are drastic enough to account for the ecological transition from a free-living to host-dependent lifestyle. The decrease in network robustness across different metabolic systems may be associated with the progressive integration in the more stable environment provided by the insect host. Finally, reductive evolution simulations reveal the strong influence that external conditions exert on the evolvability of metabolic systems.     

Design and synthesis of potent hydroxyethylamine (HEA) BACE-1 inhibitors carrying prime side 4,5,6,7-tetrahydrobenzazole and 4,5,6,7-tetrahydropyridinoazole templates

A set of low molecular weight compounds containing a hydroxyethylamine (HEA) core structure with different prime side alkyl substituted 4,5,6,7-tetrahydrobenzazoles and one 4,5,6,7-tetrahydropyridinoazole was synthesized. Striking differences were observed on potencies in the BACE-1 enzymatic and cellular assays depending on the nature of the heteroatoms in the bicyclic ring, from the low active compound 4 to inhibitor 6, displaying BACE-1 IC₅₀ values of 44 nM (enzyme assay) and 65 nM (cell-based assay).