Nuclear and chloroplast DNA phylogeography reveals Pleistocene divergence and subsequent secondary contact of two genetic lineages of the tropical rainforest tree species Shorea leprosula (Dipterocarpaceae) in South‐East Asia

Tropical rainforests in South‐East Asia have been affected by climatic fluctuations during past glacial eras. To examine how the accompanying changes in land areas and temperature have affected the genetic properties of rainforest trees in the region, we investigated the phylogeographic patterns of a widespread dipterocarp species, Shorea leprosula. Two types of DNA markers were used: expressed sequence tag‐based simple sequence repeats and chloroplast DNA (cpDNA) sequence variations. Both sets of markers revealed clear genetic differentiation between populations in Borneo and those in the Malay Peninsula and Sumatra (Malay/Sumatra). However, in the south‐western part of Borneo, genetic admixture of the lineages was observed in the two marker types. Coalescent simulation based on cpDNA sequence variation suggested that the two lineages arose 0.28–0.09 million years before present and that following their divergence migration from Malay/Sumatra to Borneo strongly exceeded migration in the opposite direction. We conclude that the genetic structure of S. leprosula was largely formed during the middle Pleistocene and was subsequently modified by eastward migration across the subaerially exposed Sunda Shelf.     

The kinetic deuterium isotope effect as applied to metabolic deactivation of imatinib to the des-methyl metabolite,CGP74588

There has recently been a burgeoning interest in impeding drug metabolism by replacing hydrogen atoms with deuterium to invoke a kinetic isotope effect. Imatinib, a front-line therapy for both chronic myeloid leukemia and of gastrointestinal stromal tumours, is often substantially metabolised via N-demethylation to the significantly less active CGP74588. Since deuterium–carbon bonds are stronger than hydrogen–carbon bonds, we hypothesised that the N-trideuteromethyl analogue of imatinib might be subject to a reduced metabolic turnover as compared to imatinib and lead to different pharmacokinetic properties, and hence improved efficacy, in vivo. Consequently, we investigated whether the N-trideuteromethyl analogue would maintain target inhibition and show a reduced propensity for N-demethylation in in vitro assays with liver microsomes and following oral administration to rats. The N-trideuteromethyl compound exhibited similar activity as a tyrosine kinase inhibitor as imatinib and similar efficacy as an antiproliferative in cellular assays. In comparison to imatinib, the trideuterated analogue also showed reduced N-demethylation upon incubation with both rat and human liver microsomes, consistent with a deuterium isotope effect. However, the reduced in vitro metabolism did not translate into increased exposure of the N-trideuteromethyl analogue following intravenous administration of the compound to rats and no significant difference was observed for the formation of the N-desmethyl metabolite from either parent drug.     

Causes and effects of haploinsufficiency

Haploinsufficiency is a form of genetic dominance and is the underlying mechanism of numerous human inherited conditions in which the causal genes are sensitive to altered dosage. This review examines the poorly understood relationships between haploinsufficiency, dosage sensitivity and genetic dominance, whose common theme is the existence of nonlinear relationships between genotype and phenotype. We present an up‐to‐date account of the bases of haploinsufficiency from the perspective of theoretical and experimental models. We also discuss human conditions caused by haploinsufficiency, including developmental syndromes and cancer. Connections between the understanding of these conditions' genetic mechanisms and advances in treatments are also described.     

Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of ∼100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.     

Frequently made mistakes in electrophoresis

In spite of text books, instrument manuals, product instructions, and web tutorials there are a number of erroneous protocols around, which lead repeatedly to issues during electrophoresis runs and to inadequate results. The relatively low resolution and short running time of miniformat systems often conceals these issues. However, in high-resolution 2-D electrophoresis in large format gels, one of the most important separation methods in Proteomics, the consequences of these mistakes become more obvious.     

Development of a bioassay to assess resistance to Fusarium oxysporum (Schlecht.) in asparagus (Asparagus officinalis L.)

Fusarium oxysporum is one of the major pathogens causing root and crown rot in asparagus. Breeding of cultivars resistant to F. oxysporum would be the most efficient strategy for pathogen control. In this study, a bioassay was developed for screening seedling resistance. The non‐destructive bioassay comprises inoculation with a highly aggressive F. oxysporum isolate, incubation in a climate chamber and quantification of disease symptoms by a digital image analysing system and a PTA‐ELISA. This bioassay is simple to implement and demonstrated high reproducibility. Subsequently, it was used to determine the resistance behaviour of 16 asparagus genotypes to F. oxysporum. The asparagus cultivars revealed different levels of susceptibility, whereas the wild relative A. densiflorus was confirmed to be resistant.     

Differential expression of the human glucocerebrosidase-coding gene

Gaucher disease is an inborn error of sphingolipid metabolism. It is due to decreased enzymatic activity of glucocerebrosidase (GCase) which causes accumulation of glucocerebrosides, mainly in cells of the reticulo-endothelial system. The disorder is clinically heterogenous and can include central nervous system signs. However, the manifestations of the disease in most cases are restricted to a limited number of cell types and organs. This could be explained by highly differential expression of the human gcs gene. To test this notion, the level of GCase-specific mRNA was determined in different human cell lines by hybridizing Northern blots to a human GCase-specific cDNA probe or by using the RNase protection method. It was found that epithelial cells exhibit high levels of GCase mRNA while skin fibroblasts and promyelocytes show intermediate steady-state levels of this RNA. Macrophages have low steady-state levels of GCase mRNA and in B-cells it is hardly detectable. Moreover, when B-cells or skin fibroblasts were transfected with a vector harbouring the bacterial cat gene coupled to the human gcs gene promoter, the levels of CAT expressed in each cell type were directly correlated to the amount of endogenous GCase RNA. Comparison of the GCase mRNA levels in Gaucher-versus non-Gaucher-derived cells revealed that in Gaucher cells this RNA is always more abundant than in the corresponding non-Gaucher counterparts, suggesting the involvement of a feed-back mechanism sensitive to the levels of actual enzymatic activity.