What have proteomics taught us about Leishmania development?

Leishmania are obligatory intracellular parasitic protozoa that cycle between sand fly mid-gut and phagolysosomes of mammalian macrophages. They have developed genetically programmed changes in gene and protein expression that enable rapid optimization of cell function according to vector and host environments. During the last two decades, host-free systems that mimic intra-lysosomal environments have been devised in which promastigotes differentiate into amastigotes axenically. These cultures have facilitated detailed investigation of the molecular mechanisms underlying Leishmania development inside its host. Axenic promastigotes and amastigotes have been subjected to transcriptome and proteomic analyses. Development had appeared somewhat variable but was revealed by proteomics to be strictly coordinated and regulated. Here we summarize the current understanding of Leishmania promastigote to amastigote differentiation, highlighting the data generated by proteomics

Crossover from percolation to diffusion

A problem of the crossover from percolation to diffusion transport is considered. A general scaling theory is proposed. It introduces phenomenologically four critical exponents which are connected by two equations. One exponent is completely new. It describes the increase of the diffusion below percolation threshold. As an example, an exact solution of one dimensional lattice problem is given. In this case the new exponent $q=2$.Comment: 10 pages, 1 figur

On the Importance of Countergradients for the Development of Retinotopy: Insights from a Generalised Gierer Model

During the development of the topographic map from vertebrate retina to superior colliculus (SC), EphA receptors are expressed in a gradient along the nasotemporal retinal axis. Their ligands, ephrin-As, are expressed in a gradient along the rostrocaudal axis of the SC. Countergradients of ephrin-As in the retina and EphAs in the SC are also expressed. Disruption of any of these gradients leads to mapping errors. Gierer's (1981) model, which uses well-matched pairs of gradients and countergradients to establish the mapping, can account for the formation of wild type maps, but not the double maps found in EphA knock-in experiments. I show that these maps can be explained by models, such as Gierer's (1983), which have gradients and no countergradients, together with a powerful compensatory mechanism that helps to distribute connections evenly over the target region. However, this type of model cannot explain mapping errors found when the countergradients are knocked out partially. I examine the relative importance of countergradients as against compensatory mechanisms by generalising Gierer's (1983) model so that the strength of compensation is adjustable. Either matching gradients and countergradients alone or poorly matching gradients and countergradients together with a strong compensatory mechanism are sufficient to establish an ordered mapping. With a weaker compensatory mechanism, gradients without countergradients lead to a poorer map, but the addition of countergradients improves the mapping. This model produces the double maps in simulated EphA knock-in experiments and a map consistent with the Math5 knock-out phenotype. Simulations of a set of phenotypes from the literature substantiate the finding that countergradients and compensation can be traded off against each other to give similar maps. I conclude that a successful model of retinotopy should contain countergradients and some form of compensation mechanism, but not in the strong form put forward by Gierer

EphA3 Expressed in the Chicken Tectum Stimulates Nasal Retinal Ganglion Cell Axon Growth and Is Required for Retinotectal Topographic Map Formation

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis

A Multi-Component Model of the Developing Retinocollicular Pathway Incorporating Axonal and Synaptic Growth

During development, neurons extend axons to different brain areas and produce stereotypical patterns of connections. The mechanisms underlying this process have been intensively studied in the visual system, where retinal neurons form retinotopic maps in the thalamus and superior colliculus. The mechanisms active in map formation include molecular guidance cues, trophic factor release, spontaneous neural activity, spike-timing dependent plasticity (STDP), synapse creation and retraction, and axon growth, branching and retraction. To investigate how these mechanisms interact, a multi-component model of the developing retinocollicular pathway was produced based on phenomenological approximations of each of these mechanisms. Core assumptions of the model were that the probabilities of axonal branching and synaptic growth are highest where the combined influences of chemoaffinity and trophic factor cues are highest, and that activity-dependent release of trophic factors acts to stabilize synapses. Based on these behaviors, model axons produced morphologically realistic growth patterns and projected to retinotopically correct locations in the colliculus. Findings of the model include that STDP, gradient detection by axonal growth cones and lateral connectivity among collicular neurons were not necessary for refinement, and that the instructive cues for axonal growth appear to be mediated first by molecular guidance and then by neural activity. Although complex, the model appears to be insensitive to variations in how the component developmental mechanisms are implemented. Activity, molecular guidance and the growth and retraction of axons and synapses are common features of neural development, and the findings of this study may have relevance beyond organization in the retinocollicular pathway

Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host

This work was supported by a Wellcome Trust Programme grant to KM and by a Wellcome Trust Strategic award to the Centre for Immunity, Infection and Evolution at the University of Edinburgh. SM was supported by a studentship from the Medical Research Council, UK.The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.Publisher PDFPeer reviewe

What have proteomics taught us about Leishmania development?

Leishmania are obligatory intracellular parasitic protozoa that cycle between sand fly mid-gut and phagolysosomes of mammalian macrophages. They have developed genetically programmed changes in gene and protein expression that enable rapid optimization of cell function according to vector and host environments. During the last two decades, host-free systems that mimic intra-lysosomal environments have been devised in which promastigotes differentiate into amastigotes axenically. These cultures have facilitated detailed investigation of the molecular mechanisms underlying Leishmania development inside its host. Axenic promastigotes and amastigotes have been subjected to transcriptome and proteomic analyses. Development had appeared somewhat variable but was revealed by proteomics to be strictly coordinated and regulated. Here we summarize the current understanding of Leishmania promastigote to amastigote differentiation, highlighting the data generated by proteomics

Isotopic composition of uranium in soil and ground water samples collected around 30 Km Chernobyl exclusion zone

日本放射線影響学会第４９回大

Isotopic Ratios of Uranium in Soil and Ground Water Samples collected around 30 Km Chernobyl Exclusion Zone

Precise and accurate measurement uranium isotope ratio is essential in environmental monitoring of any contamination in nuclear safeguards. 238U, 235U and 234U are naturally occurring alpha-emitting long-lived radionuclides which are taken up daily at low levels with food and drinking. IUPAC has established natural isotopic composition of 235U/238U to be 0.00725. Therefore, isotope ratio measurements are important because they can provide information on the origin of uranium. The isotope ratios of uranium, 234U/238U, 235U/238U and 236U/238U were measured using a VG Sector 54 thermal ionization mass spectrometer (TIMS) as well as high resolution inductively coupled plasma mass spectrometry (ICP-MS) in soil samples as well as in some water samples collected in the exclusion zone of Chernobyl nuclear power plant. The isotopic composition of Chernobyl soil samples showed significant deviation from the natural uranium and presence of 236U is quite noticeable. The 234U/238U activity ratio varies in the range of 1.06 to 2.1 and 1.42 to 5.75 for soil and water samples, respectively. Enrichment of 235U was also noticeable for soil as well as ground water samples.International Conference on Radioecology & Environmental Radioactivit

Regulation dynamics of Leishmania differentiation: deconvoluting signals and identifying phosphorylation trends

Leishmania are obligatory intracellular parasitic protozoa that cause a wide range of disease in humans; cycling between extracellular promastigotes in the mid-gut of sand flies and intracellular amastigotes in the phagolysosome of mammalian macrophages. While much of the molecular mechanism of development inside macrophages remains a mystery, development of a host-free system that simulates phagolysosome conditions (37ºC and pH 5.5) has provided new insights into these processes. The time-course of promastigote-to-amastigote differentiation can be divided into four morphologically distinct phases: I, signal perception (0-5 hours after exposure); II, movement cessation and aggregation (5-10 hours); III, amastigote morphogenesis (10-24 hours); and IV, maturation (24-120 hours). Transcriptomic and proteomic analyses indicated that differentiation is a coordinated process that results in adaptation to life inside phagolysosomes. Recent phosphoproteomic analysis revealed extensive differences in phosphorylation between promastigotes and amastigotes, and identified stage-specific phosphorylation motifs. We hypothesize that the differentiation signal activates a phosphorylation pathway, which initiates Leishmania transformation, and here we use Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) to interrogate the dynamics of changes in phosphorylation profile during Leishmania donovani promastigote-to-amastigote differentiation. Analysis of 163 phosphopeptides (from 106 proteins) revealed six distinct kinetic profiles; with increases in phosphorylation predominating during phase I and III, whereas phase II and IV are characterized by greater dephosphorylation. Several proteins (including a protein kinase) were phosphorylated in phase I after exposure to the complete differentiation signal (i.e. signalspecific; 37ºC and pH 5.5); but not after either of the physical parameters separately. Several other protein kinases (including regulatory subunits) and phosphatases also showed changes in phosphorylation during differentiation. This work constitutes the first genome-scale interrogation of phosphorylation dynamics in a parasitic protozoa; revealing the outline of a signaling pathway during Leishmania differentiation. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (identifier PXD000671). Data can be viewed using ProteinPilot™ software: http://www.absciex.com/products/software/proteinpilot-software