Ubiquitination of the glycosomal matrix protein receptor PEX5 in Trypanosoma brucei by PEX4 displays novel features

Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ?PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ?PEX4 mutant.     

Notch-signalling is required for head regeneration and tentacle patterning in Hydra

Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals.     

Mathematical Modeling of Respiratory System Mechanics in the Newborn Lamb

In this paper, a mathematical model of the respiratory mechanics is used to reproduce experimental signal waveforms acquired from three newborn lambs. As the main challenge is to determine specific lamb parameters, a sensitivity analysis has been realized to find the most influent parameters, which are identified using an evolutionary algorithm. Results show a close match between experimental and simulated pressure and flow waveforms obtained during spontaneous ventilation and pleural pressure variations acquired during the application of positive pressure, since root mean square errors equal to 0.0119, 0.0052 and 0.0094. The identified parameters were discussed in light of previous knowledge of respiratory mechanics in the newborn.     

Effects of surfactin on membrane models displaying lipid phase separation

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.     

A fast growing spectrum of biological functions of γ-secretase in development and disease

γ-secretase, which assembles as a tetrameric complex, is an aspartyl protease that proteolytically cleaves substrate proteins within their membrane-spanning domain; a process also known as regulated intramembrane proteolysis (RIP). RIP regulates signaling pathways by abrogating or releasing signaling molecules. Since the discovery, already > 15 years ago, of its catalytic component, presenilin, and even much earlier with the identification of amyloid precursor protein as its first substrate, γ-secretase has been commonly associated with Alzheimer's disease. However, starting with Notch and thereafter a continuously increasing number of novel substrates, γ-secretase is becoming linked to an equally broader range of biological processes. This review presents an updated overview of the current knowledge on the diverse molecular mechanisms and signaling pathways controlled by γ-secretase, with a focus on organ development, homeostasis and dysfunction. This article is part of a Special Issue entitled: Intramembrane Proteases.     

A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de‐vernalization responses

Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT‐PCR and named FLC‐LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over‐expression of CiFL1 in Arabidopsis caused late flowering and prevented up‐regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post‐vernalization temperature was favorable to flowering and when it de‐vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re‐activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold‐induced down‐regulation of a MADS box floral repressor and its re‐activation by high temperature thus appear to be conserved features of the vernalization and de‐vernalization responses in distant species.     

Substituted imidazopyridazines are potent and selective inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1)

A series of imidazopyridazines which are potent inhibitors of Plasmodium falciparum calcium-dependent protein kinase 1 (PfCDPK1) was identified from a high-throughput screen against the isolated enzyme. Subsequent exploration of the SAR and optimisation has yielded leading members which show promising in vitro anti-parasite activity along with good in vitro ADME and selectivity against human kinases. Initial in vivo testing has revealed good oral bioavailability in a mouse PK study and modest in vivo efficacy in a Plasmodium berghei mouse model of malaria.     

Imidazo[4,5-b]pyridine inhibitors of B-Raf kinase

This Letter details the synthesis and evaluation of imidazo[4,5-b]pyridines as inhibitors of B-Raf kinase. These compounds bind in a DFG-in, αC-helix out conformation of B-Raf, which is a binding mode associated with significant kinase selectivity. Structure–activity relationship studies involved optimization of the ATP-cleft binding region of these molecules, and led to compound 23, an inhibitor with excellent enzyme/cell potency, and kinase selectivity.     

Eucalypts face increasing climate stress

Global climate change is already impacting species and ecosystems across the planet. Trees, although long‐lived, are sensitive to changes in climate, including climate extremes. Shifts in tree species' distributions will influence biodiversity and ecosystem function at scales ranging from local to landscape; dry and hot regions will be especially vulnerable. The Australian continent has been especially susceptible to climate change with extreme heat waves, droughts, and flooding in recent years, and this climate trajectory is expected to continue. We sought to understand how climate change may impact Australian ecosystems by modeling distributional changes in eucalypt species, which dominate or codominate most forested ecosystems across Australia. We modeled a representative sample of Eucalyptus and Corymbia species (n = 108, or 14% of all species) using newly available Representative Concentration Pathway (RCP) scenarios developed for the 5th Assessment Report of the IPCC, and bioclimatic and substrate predictor variables. We compared current, 2025, 2055, and 2085 distributions. Overall, Eucalyptus and Corymbia species in the central desert and open woodland regions will be the most affected, losing 20% of their climate space under the mid‐range climate scenario and twice that under the extreme scenario. The least affected species, in eastern Australia, are likely to lose 10% of their climate space under the mid‐range climate scenario and twice that under the extreme scenario. Range shifts will be lateral as well as polewards, and these east–west transitions will be more significant, reflecting the strong influence of precipitation rather than temperature changes in subtropical and midlatitudes. These net losses, and the direction of shifts and contractions in range, suggest that many species in the eastern and southern seaboards will be pushed toward the continental limit and that large tracts of currently treed landscapes, especially in the continental interior, will change dramatically in terms of species composition and ecosystem structure.     

Single‐nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula

Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single‐nucleotide polymorphism (SNP) markers in M. truncatula through large‐scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina's GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.