Cathepsin L silencing enhances arsenic trioxide mediated in vitro cytotoxicity and apoptosis in glioblastoma U87MG spheroids

Despite improved treatment options, glioblastoma multiforme (GBM) remains the most aggressive brain tumour with the shortest post-diagnostic survival. Arsenite (As₂O₃) is already being used in the treatment of acute promyelocytic leukaemia (APL), yet its effects on GBM have not been evaluated in detail. In U87MG cell monolayers, we have previously shown that arsenite cytotoxicity significantly increases upon transient inhibition of lysosomal protease Cathepsin L (CatL). As multicellular spheroids more closely represent in vivo tumours, we aimed to evaluate the impact of permanent CatL silencing on arsenite treatment in U87MG spheroids. CatL was stably silenced using shRNA expression plasmid packed lentiviruses. By using metabolic- and cell viability assays, we demonstrated that long-term CatL silencing significantly increased arsenite cytotoxicity in U87MG spheroids. Silenced CatL also increased arsenite-mediated apoptosis in spheroids via elevated p53 expression, Bax/Bcl2 ratio and caspase 3/7 activity, though with lower efficacy than in monolayers. Arsenite cytotoxicity was enhanced by lower CatL activity, since similar cytotoxicity increase was also observed using the novel CatL inhibitor AT094. The results have significant translational impact, since stable CatL silencing would enable the application of lower systemic doses of arsenite to achieve the desired cytotoxic effects on GBMs in vivo.     

Automated Masking of AFLP Markers Improves Reliability of Phylogenetic Analyses

The amplified fragment length polymorphisms (AFLP) method has become an attractive tool in phylogenetics due to the ease with which large numbers of characters can be generated. In contrast to sequence-based phylogenetic approaches, AFLP data consist of anonymous multilocus markers. However, potential artificial amplifications or amplification failures of fragments contained in the AFLP data set will reduce AFLP reliability especially in phylogenetic inferences. In the present study, we introduce a new automated scoring approach, called “AMARE” (AFLP MAtrix REduction). The approach is based on replicates and makes marker selection dependent on marker reproducibility to control for scoring errors. To demonstrate the effectiveness of our approach we record error rate estimations, resolution scores, PCoA and stemminess calculations. As in general the true tree (i.e. the species phylogeny) is not known, we tested AMARE with empirical, already published AFLP data sets, and compared tree topologies of different AMARE generated character matrices to existing phylogenetic trees and/or other independent sources such as morphological and geographical data. It turns out that the selection of masked character matrices with highest resolution scores gave similar or even better phylogenetic results than the original AFLP data sets.     

Copy number determination of different derivatives of the streptomycete mini-plasmid pSLG33

Copy numbers of the streptomycete plasmid vector pRS410 and five other recombinant plasmid derivatives of the original cryptic streptomycete plasmid pSLG33 were determined using calibrated laser densitometry. DNA preparations, electrophoretically separated on agarose gels, were stained with ethidium bromide, photographed and the negatives were subsequently scanned in a laser densitometer. The pSLG33 replicon is very stable, as no effect of the selective pressure was observed. It is a multicopy plasmid with up to 220 detected copies per chromosome. The use of deletion and/or insertion mutants allowed us to define two regions of the pSLG33 molecule involved in the control of plasmid replication.     

Palaeoecological and phytosociological reconstruction of precultural vegetation in the Bílý Kříž area,the Moravskoslezské Beskydy Mts,Slovak Republic

A combined palynological and phytosociological methodological approach was used in the reconstruction of precultural forest vegetation in a small model area of Bílý Kí-Janík in the Moravskoslezské Beskydy mountains (northwest Slovakia). Two pollen diagrams show the predominance of Fagus sylvatica, Abies alba and Picea abies in past natural mixed forests. The present-day occurrence and distribution of forest herbs allow the differentiation of five past vegetation types with above mentioned forest climax trees covering the area before the beginning of human interference. A map of reconstructed natural vegetation is presented which can be used for revitalization of present secondary spruce plantations, heavily affected by air pollution.     

Insect pest management in the age of synthetic biology

Arthropod crop pests are responsible for 20% of global annual crop losses, a figure predicted to increase in a changing climate where the ranges of numerous species are projected to expand. At the same time, many insect species are beneficial, acting as pollinators and predators of pest species. For thousands of years, humans have used increasingly sophisticated chemical formulations to control insect pests but, as the scale of agriculture expanded to meet the needs of the global population, concerns about the negative impacts of agricultural practices on biodiversity have grown. While biological solutions, such as biological control agents and pheromones, have previously had relatively minor roles in pest management, biotechnology has opened the door to numerous new approaches for controlling insect pests. In this review, we look at how advances in synthetic biology and biotechnology are providing new options for pest control. We discuss emerging technologies for engineering resistant crops and insect populations and examine advances in biomanufacturing that are enabling the production of new products for pest control.     

Repeat proliferation and partial endoreplication jointly shape the patterns of genome size evolution in orchids

Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2–5.1 Gbp) was mostly independent of profound chromosome count variation (2n = 12–90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated in the non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape the patterns of genome size diversity in orchids.