Dynamic RNA profiling in Plasmodium falciparum synchronized blood stages exposed to lethal doses of artesunate

<p>Abstract</p> <p>Background</p> <p>Translation of the genome sequence of <it>Plasmodium sp</it>. into biologically relevant information relies on high through-put genomics technology which includes transcriptome analysis. However, few studies to date have used this powerful approach to explore transcriptome alterations of <it>P. falciparum </it>parasites exposed to antimalarial drugs.</p> <p>Results</p> <p>The rapid action of artesunate allowed us to study dynamic changes of the parasite transcriptome in synchronous parasite cultures exposed to the drug for 90 minutes and 3 hours. Developmentally regulated genes were filtered out, leaving 398 genes which presented altered transcript levels reflecting drug-exposure. Few genes related to metabolic pathways, most encoded chaperones, transporters, kinases, Zn-finger proteins, transcription activating proteins, proteins involved in proteasome degradation, in oxidative stress and in cell cycle regulation. A positive bias was observed for over-expressed genes presenting a subtelomeric location, allelic polymorphism and encoding proteins with potential export sequences, which often belonged to subtelomeric multi-gene families. This pointed to the mobilization of processes shaping the interface between the parasite and its environment. In parallel, pathways were engaged which could lead to parasite death, such as interference with purine/pyrimidine metabolism, the mitochondrial electron transport chain, proteasome-dependent protein degradation or the integrity of the food vacuole.</p> <p>Conclusion</p> <p>The high proportion of over-expressed genes encoding proteins exported from the parasite highlight the importance of extra-parasitic compartments as fields for exploration in drug research which, to date, has mostly focused on the parasite itself rather than on its intra and extra erythrocytic environment. Further work is needed to clarify which transcriptome alterations observed reflect a specific response to overcome artesunate toxicity or more general perturbations on the path to cellular death.</p

A micro-bead device to explore Plasmodium falciparum-infected, spherocytic or aged red blood cells prone to mechanical retention by spleen endothelial slits

Présentation oraleInternational audiencen.

Computational methodology to determine fluid related parameters on non regular three-dimensional scaffolds

The application of three-dimensional (3D) biomaterials to facilitate the adhesion, proliferation, and differentiation of cells has been widely studied for tissue engineering purposes. The fabrication methods used to improve the mechanical response of the scaffold produce complex and non regular structures. Apart from the mechanical aspect, the fluid behavior in the inner part of the scaffold should also be considered. Parameters such as permeability (k) or wall shear stress (WSS) are important aspects in the provision of nutrients, the removal of metabolic waste products or the mechanically-induced differentiation of cells attached in the trabecular network of the scaffolds. Experimental measurements of these parameters are not available in all labs. However, fluid parameters should be known prior to other types of experiments. The present work compares an experimental study with a computational fluid dynamics (CFD) methodology to determine the related fluid parameters (k and WSS) of complex non regular poly(L-lactic acid) scaffolds based only on the treatment of microphotographic images obtained with a microCT (lCT). The CFD analysis shows similar tendencies and results with low relative difference compared to those of the experimental study, for high flow rates. For low flow rates the accuracy of this prediction reduces. The correlation between the computational and experimental results validates the robustness of the proposed methodology.The authors gratefully acknowledge research support from the Spanish Ministry of Science and Innovation through research project DPI2010-20399-C04-01. The Instituto de Salud Carlos III (ISCIII) through the CIBER initiative and the Platform for Biological Tissue Characterization of the Centro de Investigacion Biomedica en Red en Bioingenieria, Biomateriales y Nanomedicina (CIBER-BBN) are also gratefully acknowledged.Acosta Santamaría, VA.; Malvé, M.; Duizabo, A.; Mena Tobar, A.; Gallego Ferrer, G.; García Aznar, J.; Doblare Castellano, M.... (2013). Computational methodology to determine fluid related parameters on non regular three-dimensional scaffolds. Annals of Biomedical Engineering. 41(11):2367-2380. https://doi.org/10.1007/s10439-013-0849-8S236723804111Acosta Santamaría, V., H. Deplaine, D. Mariggió, A. R. Villanueva-Molines, J. M. García-Aznar, J. L. Gómez Ribelles, M. Doblaré, G. Gallego Ferrer, and I. Ochoa. Influence of the macro and micro-porous structure on the mechanical behavior of poly(l-lactic acid) scaffolds. J. Non-Cryst. Solids 358(23):3141–3149, 2012.Adachi, T., Y. Osako, M. Tanaka, M. Hojo, and S. J. Hollister. 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Vers une étude de la résistance de P.falciparum à l'artésunate (identification d'un gène impliqué dans la tolérance du parasite à l'arténusate)

L émergence de résistances aux artémisinines in vitro met en danger le traitement par ACT (Artemisinin-based combined treatments). Caractérisation d'un gène modulant la tolérance de P.falciparum à l'artésunate : L étude du transcriptome sous artésunate montre une modification de l expression de près de 400 gènes. Pf10_0026, le plus surexprimé, est de fonction non prédite. Il appartient à une nouvelle famille, spécifique aux Plasmodies, caractérisée par un domaine structuré riche en tryptophane. PF10_0026 i) augmente sous artésunate de façon dose-dépendante ii) est localisée dans le cytoplasme parasitaire. La surexpression de Pf10_0026 confère une tolérance à l artésunate.Substitut in vitro au filtre splénique : La rate joue un rôle-clé dans l élimination des GRP traités par l artésunate. Les GR subissent des déformations majeures pour traverser les fentes inter-endothéliales (FIE). Un substitut in vitro au filtre splénique a été mis au point grâce à une filtration sur des billes de diamètre contrôlé. Ce modèle a été validé sur des GR de déformabilité réduite (traitement à 50C, GR sphérocytaires) puis comparé au système de rate isolée-perfuséePARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Quantitative assessment of sensing and sequestration of spherocytic erythrocytes by the human spleen

Introduction During their 120-day life span, human RBCs repeatedly traverse capillaries of the vascular bed and interendothelial slits of the venous sinus of spleen red pulp, both of which are narrower than their smallest dimension. 1 This necessitates maintenance of the ability of RBCs to undergo repeated, extensive, and reversible deformations. Repeated major membrane deformations induce ion and water permeability changes in the RBCs. Although a major role for the spleen in pathogenesis of HS is well established, RBCs with defined loss in membrane surface area can be generated experimentally by treatment with lysophosphatidylcholine (LPC). 6,7 By initially accumulating exclusively in the external leaflet of the RBC lipid bilayer, LPC induces in a dose-dependent manner echinocytosis There is an Inside Blood commentary on this article in this issue. The online version of this article contains a data supplement. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked &apos;&apos;advertisement&apos;&apos; in accordance with 18 USC section 1734. For personal use only. on October 7, 2016. by guest www.bloodjournal.org From (spiculation) and eventually spherocytes through the release of microvesicles. Methods This study was approved by the investigational review board of Ile-deFrance II (Paris, France). Human spleen retrieval Spleens were retrieved and processed as described previously. RBC labeling with PKH67 and/or PKH26 Blood from a blood center (Etablissement Français du Sang, Rungis, France) was washed 3 times in RPMI 1640 to remove WBCs. RBCs were labeled with the lipophilic fluorescent probes PKH67 or PKH26 (hematocrit, 5%) according to the directions of the manufacturer (Sigma-Aldrich) with some modifications to obtain 4 different populations of labeled RBCs. Two populations of RBCs were labeled with one PKH (RBC/PKH67, PKH67 dilution 1/1000; RBC/PKH26, PKH26 dilution 1/250). Two other populations of RBCs were labeled with both PKHs at different concentrations (RBC/KH26-67, PKH67 dilution 1/2000 plus PKH26 dilution 1/500; RBC/PKH67-26, PKH67 dilution 1/1000 plus PKH26 dilution 1/3000). These 4 populations of labeled RBCs could be distinguished by FACS, which allowed us to perfuse 4 distinct preparations in a single spleen and trace each population individually. Treatment of RBCs with LPC PKH-labeled RBCs were resuspended in LPC (0-15M) in PBS or PBS alone at a 1% hematocrit level to induce a controlled loss of membrane area. The LPC samples were incubated for 5 minutes at room temperature. After incubation, samples were washed 3 times with PBS and resuspended in Krebs-albumin for further analysis. Measurement of RBC deformability RBC deformability was measured by ektacytometry using a laser-assisted optical rotational cell analyzer (Mechatronics) as described previously. Ex vivo spleen perfusion Isolated-perfused spleen experiments were performed as described previously 20,25 in a Plexiglas chamber maintained at 37°C by a regulated warm air flow. The perfusion of mixture of untreated (RBC/PKH) or LPC-treated (RBC/PKH/LPC) PKH-labeled RBCs (15%-25% final hematocrit in Krebsalbumin medium) was performed over a 1-to 2-hour period. The percentage of circulating LPC-treated cells was quantified by flow cytometric analysis (FACSCalibur; BD Biosciences). Data were analyzed using CellQuest 3.3 software (BD Biosciences). Analysis of RBC morphology and dimensions Untreated or LPC-treated RBCs were fixed with PBS-paraformaldehyde (1%) for analysis. Image acquisition and data analysis were done as described previously. Osmotic fragility test Osmotic fragility of RBCs was determined according to the method originally described by Parpart et al. Scanning electron microscopy RBC specimens were fixed in 2.5% glutaraldehyde in cacodylate buffer (0.1M) overnight at 4°C. RBCs were dropped gently onto a glass coverslip that had been treated with polylysine. After 3 cacodylate buffer rinses (10 minutes each), the specimens were fixed with 1% osmium tetroxide for 1 hour. After 3 rapid washes in water, samples were processed through an ethanol dehydration series of 25%-100% ethanol, followed by critical point drying with CO 2 . Dried specimens were sputtered with 22-nm gold palladium, examined, and photographed with a JEOL JSM 6700F field emission scanning electron microscope operating at 5 or 7 kV. Images were acquired with the upper detector and the lower secondary detector. Results RBC treatment with LPC results in a dose-dependent loss of surface area and deformability RBCs were exposed to increasing LPC concentrations to generate RBC populations with progressively decreasing membrane surface areas ( SURFACE-DEFICIENT RBCs AND THE HUMAN SPLEEN 425 BLOOD, 12 JULY 2012 ⅐ VOLUME 120, NUMBER 2 For personal use only. on October 7, 2016. by guest www.bloodjournal. org From Ektacytometric analysis showed that LPC-treated RBCs exhibited lower cellular deformability than control RBCs Retention of LPC-treated RBCs by the isolated-perfused human spleen is determined by the extent of surface area loss To understand how the human spleen handles RBCs displaying decreased surface area at constant mean cell volume (ie, with a Adaptation of a subset of surface-deficient RBCs during splenic transit Even at the highest extent of membrane surface area loss, a subset of LPC-treated RBCs exited from the spleen, as reflected by the existence of a plateau in cell clearance studies ( SURFACE-DEFICIENT RBCs AND THE HUMAN SPLEEN 427 BLOOD, 12 JULY 2012 ⅐ VOLUME 120, NUMBER 2 For personal use only. on October 7, 2016. by guest www.bloodjournal.org From volume enabled the sustained circulation of this population of cells with reduced surface area. Discussion The results of the present study demonstrate clearly that cell surface area loss with reduced S/V ratio is a significant predictor of splenic sequestration of human RBCs. A loss of 18% of the cell surface area (corresponding to a Ͼ 27% decrease of the S/V ratio) led to rapid RBC entrapment in normal human spleens. The majority of these surface-deprived RBCs were trapped during their first passage through the isolated spleen. Therefore, it appears that their spheroidal shape renders them incapable of undergoing the cellular deformation necessary to squeeze across the interendothelial slits of the venous sinus, which have apertures of 0.2-0.5 ϫ 2-3 m. In the present study, we found that even at the highest extent of membrane surface area loss, a subset of LPC-treated RBCs exited from the spleen. Several hypotheses might explain these results. First, the heterogeneity of the initial population of LPC-treated RBCs might include more spherical RBCs (preferentially entrapped in the 428 SAFEUKUI et al BLOOD, 12 JULY 2012 ⅐ VOLUME 120, NUMBER 2 For personal use only. on October 7, 2016. by guest www.bloodjournal.org From spleen) and LPC-treated RBCs with dimensions closer to normal values. The fact that LPC-treated RBC dimensions (for all concentrations of LPC) were similar between T0 and T40 and were both reduced significantly from that of untreated RBC

Artesunate Tolerance in Transgenic Plasmodium falciparum Parasites Overexpressing a Tryptophan-Rich Protein▿†

Due to their rapid, potent action on young and mature intraerythrocytic stages, artemisinin derivatives are central to drug combination therapies for Plasmodium falciparum malaria. However, the evidence for emerging parasite resistance/tolerance to artemisinins in southeast Asia is of great concern. A better understanding of artemisinin-related drug activity and resistance mechanisms is urgently needed. A recent transcriptome study of parasites exposed to artesunate led us to identify a series of genes with modified levels of expression in the presence of the drug. The gene presenting the largest mRNA level increase, Pf10_0026 (PArt), encoding a hypothetical protein of unknown function, was chosen for further study. Immunodetection with PArt-specific sera showed that artesunate induced a dose-dependent increase of the protein level. Bioinformatic analysis showed that PArt belongs to a Plasmodium-specific gene family characterized by the presence of a tryptophan-rich domain with a novel hidden Markov model (HMM) profile. Gene disruption could not be achieved, suggesting an essential function. Transgenic parasites overexpressing PArt protein were generated and exhibited tolerance to a spike exposure to high doses of artesunate, with increased survival and reduced growth retardation compared to that of wild-type-treated controls. These data indicate the involvement of PArt in parasite defense mechanisms against artesunate. This is the first report of genetically manipulated parasites displaying a stable and reproducible decreased susceptibility to artesunate, providing new possibilities to investigate the parasite response to artemisinins