Intraflagellar transport speed is sensitive to genetic and mechanical perturbations to flagellar beating.

Two sets of motor proteins underpin motile cilia/flagella function. The axoneme-associated inner and outer dynein arms drive sliding of adjacent axoneme microtubule doublets to periodically bend the flagellum for beating, while intraflagellar transport (IFT) kinesins and dyneins carry IFT trains bidirectionally along the axoneme. Despite assembling motile cilia and flagella, IFT train speeds have only previously been quantified in immobilized flagella-mechanical immobilization or genetic paralysis. This has limited investigation of the interaction between IFT and flagellar beating. Here, in uniflagellate Leishmania parasites, we use high-frequency, dual-color fluorescence microscopy to visualize IFT train movement in beating flagella. We discovered that adhesion of flagella to a microscope slide is detrimental, reducing IFT train speed and increasing train stalling. In flagella free to move, IFT train speed is not strongly dependent on flagella beat type; however, permanent disruption of flagella beating by deletion of genes necessary for formation or regulation of beating showed an inverse correlation of beat frequency and IFT train speed.

Cell Cycle, Telomeres, and Telomerase in Leishmania spp.: What Do We Know So Far?

Leishmaniases belong to the inglorious group of neglected tropical diseases, presenting different degrees of manifestations severity. It is caused by the transmission of more than 20 species of parasites of the Leishmania genus. Nevertheless, the disease remains on the priority list for developing new treatments, since it affects millions in a vast geographical area, especially low-income people. Molecular biology studies are pioneers in parasitic research with the aim of discovering potential targets for drug development. Among them are the telomeres, DNA-protein structures that play an important role in the long term in cell cycle/survival. Telomeres are the physical ends of eukaryotic chromosomes. Due to their multiple interactions with different proteins that confer a likewise complex dynamic, they have emerged as objects of interest in many medical studies, including studies on leishmaniases. This review aims to gather information and elucidate what we know about the phenomena behind Leishmania spp. telomere maintenance and how it impacts the parasite's cell cycle.

Apoptosis-like cell death upon kinetoplastid induction by compounds isolated from the brown algae Dictyota spiralis.

BACKGROUND: The in vitro activity of the brown seaweed Dictyota spiralis against both Leishmania amazonensis and Trypanosoma cruzi was evaluated in a previous study. Processing by bio-guided fractionation resulted in the isolation of three active compounds, classified as diterpenes. In the present study, we performed several assays to detect clinical features associated to cell death in L. amazonensis and T. cruzi with the aim to elucidate the mechanism of action of these compounds on parasitic cells. METHODS: The aims of the experiments were to detect and evaluate specific events involved in apoptosis-like cell death in the kinetoplastid, including DNA condensation, accumulation of reactive oxygen species and changes in ATP concentration, cell permeability and mitochondrial membrane potential, respectively, in treated cells. RESULTS: The results demonstrated that the three isolated diterpenes could inhibit the tested parasites by inducing an apoptosis-like cell death. CONCLUSIONS: These results encourage further investigation on the isolated compounds as potential drug candidates against both L. amazonensis and T. cruzi.

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis.

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

Synergistic response of physicochemical reaction parameters on biogenesis of silver nanoparticles and their action against colon cancer and leishmanial cells.

Physicochemical parameters include pH, temperature, the concentration of the AgNO3, ratio of reactants, agitation and incubation period that act synergistically and provide a steering force to modulate the biogenesis of nanoparticles by influencing the molecular dynamics, reaction kinetics, protein conformations, and catalysis. The current study involved the bio-fabrication of silver nanoparticles (SNPs) by using the reducing abilities of Mentha longifolia (L.) L. leaves aqueous extract. Spectrophotometric analysis of various biochemical reactions showed that 3 mM of AgNO3 at 120 °C in an acidic pH when mixed in 1-9 ratio of plant extract and AgNO3 respectively, are the optimised conditions for SNPs synthesis. Different analytical techniques confirmed that the nanoparticles are anisotropic and nearly spherical and have a size range of 10-100 nm. The ∼10 µg/ml of SNPs killed ∼66% of Leishmania population and IC50 was measured at 8.73 µg/ml. SRB assay and Annexin V apoptosis assay results showed that the plant aqueous extract and SNPs are not active against HCT116 colon cancer cells and no IC50 (80% survival) was reported. ROS generation was quantified at 0.08 Φ, revealed that the SNPs from M. longifolia can generate free radicals and no photothermal activity was recorded which makes them non-photodynamic.

Isolation of Extracellular Vesicles from Leishmania spp.

Exosomes, a class of extracellular vesicles, are released by eukaryotes, bacteria, and archaea, as evident from both in vitro and in vivo studies. These nano-sized double-membraned vesicles play an important role in cell-to-cell communication, dysregulation of the immune system, and pathogenesis in a number of diseases, including leishmaniasis. Leishmania is a genus of obligate intracellular parasites, which infect host macrophages, are transmitted through the bite of a sandfly, and are shown to secrete exosomes with immunomodulatory activities. Given the importance of these vesicles in Leishmania spp. virulence, it is necessary to perform appropriate isolation and characterization in order to further study their relevance in the parasite's infectious life cycle. In this chapter, we describe four methods for the isolation of extracellular vesicles derived from Leishmania species including ultracentrifugation, polyethylene glycol-based precipitation, size-exclusion chromatography, and sucrose-gradient fractionation. Further, we describe the preparation of isolated samples for characterization by nanoparticle tracking analysis, transmission electron microscopy, and proteomic profiling.

Cellular Markers for the Identification of Chemoresistant Isolates in Leishmania.

Markers to diagnose chemoresistance in infecting Leishmania parasites are urgently required. This is fundamental for patients who do not heal during or after treatment, as they are unresponsive, or patients who relapse at the end of the therapy, suffering from therapeutic failure. Glucose utilization is an indicator of cell viability that closely associates with metabolic activity. In Leishmania, glucose is a source of carbon atoms and is imported into the cell through specific transporters. In experimentally developed chemoresistant Leishmania parasites a significant decrease of the expression of glucose transporters as well as in the cellular accumulation glucose has been described. Alternatively, the electrical membrane potential is an essential parameter for the formation of the electromotive force needed for the acquisition of important nutrients and solutes (e.g., glucose) by cells, and changes in glucose concentration are suggested to constitute a physiological adaptation associated with a chemoresistant phenotype of Leishmania parasites. Here we describe easy methods to measure glucose uptake and the membrane potential in isolates from patient suffering leishmaniasis. Correlation between both parameters might be helpful to identify chemoresistant parasites. Results suggest that the measured kinetics of glucose utilization rate can be correlated with the plasma membrane potential and together used to differentiate between the performance of wild-type and reference parasites on the one hand and parasites isolated from patients with therapeutic failure on the other.

Activation of artemisinin and heme degradation in Leishmania tarentolae promastigotes: A possible link.

Endoperoxides (EPs) appear to be promising drug candidates against protozoal diseases, including malaria and leishmaniasis. Previous studies have shown that these drugs need an intracellular activation to exert their pharmacological potential. The efficiency of these drugs is linked to the extensive iron demand of these intracellular protozoal parasites. An essential step of the activation mechanism of these drugs is the formation of radicals in Leishmania. Iron is a known trigger for intracellular radical formation. However, the activation of EPs by low molecular iron or by heme iron may strongly depend on the structure of the EPs themselves. In this study, we focused on the activation of artemisinin (Art) in Leishmania tarentolae promastigotes (LtP) in comparison to reference compounds. Viability assays in different media in the presence of different iron sources (hemin/fetal calf serum) showed that IC50 values of Art in LtP were modulated by assay conditions, but overall were within the low micromolar range. Low temperature electron paramagnetic resonance (EPR) spectroscopy of LtP showed that Art shifted the redox state of the labile iron pool less than the EP ascaridole questioning its role as a major activator of Art in LtP. Based on the high reactivity of Art with hemin in previous biomimetic experiments, we focused on putative heme-metabolizing enzymes in Leishmania, which were so far not well described. Inhibitors of mammalian heme oxygenase (HO; tin and chromium mesoporphyrin) acted antagonistically to Art in LtP and boosted its IC50 value for several magnitudes. By inductively coupled plasma methods (ICP-OES, ICP-MS) we showed that these inhibitors do not block iron (heme) accumulation, but are taken up and act within LtP. These inhibitors blocked the conversion of hemin to bilirubin in LtP homogenates, suggesting that an HO-like enzyme activity in LtP exists. NADPH-dependent degradation of Art and hemin was highest in the small granule and microsomal fractions of LtP. Photometric measurements in the model Art/hemin demonstrated that hemin requires reduction to heme and that subsequently an Art/heme complex (λmax 474 nm) is formed. EPR spin-trapping in the system Art/hemin revealed that NADPH, ascorbate and cysteine are suitable reductants and finally activate Art to acyl-carbon centered radicals. These findings suggest that heme is a major activator of Art in LtP either via HO-like enzyme activities and/or chemical interaction of heme with Art.

Towards discovery of new leishmanicidal scaffolds able to inhibit Leishmania GSK-3.

Previous reports have validated the glycogen synthase kinase-3 (GSK-3) as a druggable target against the human protozoan parasite Leishmania. This prompted us to search for new leishmanicidal scaffolds as inhibitors of this enzyme from our in-house library of human GSK-3ß inhibitors, as well as from the Leishbox collection of leishmanicidal compounds developed by GlaxoSmithKline. As a result, new leishmanicidal inhibitors acting on Leishmania GSK-3 at micromolar concentrations were found. These inhibitors belong to six different chemical classes (thiadiazolidindione, halomethylketone, maleimide, benzoimidazole, N-phenylpyrimidine-2-amine and oxadiazole). In addition, the binding mode of the most active compounds into Leishmania GSK-3 was approached using computational tools. On the whole, we have uncovered new chemical scaffolds with an appealing prospective in the development and use of Leishmania GSK-3 inhibitors against this infectious protozoan.

In vitro activity of 1H-phenalen-1-one derivatives against Leishmania spp. and evidence of programmed cell death.

BACKGROUND: The in vitro activity against Leishmania spp. of a novel group of compounds, phenalenone derivatives, is described in this study. Previous studies have shown that some phenalenones present leishmanicidal activity, and induce a decrease in the mitochondrial membrane potential in L. amazonensis parasites, so in order to elucidate the evidence of programmed cell death occurring inside the promastigote stage, different assays were performed in two different species of Leishmania. METHODS: We focused on the determination of the programmed cell death evidence by detecting the characteristic features of the apoptosis-like process, such as phosphatidylserine exposure, mitochondrial membrane potential, and chromatin condensation among others. RESULTS: The results showed that four molecules activated the apoptosis-like process in the parasite. All the signals observed were indicative of the death process that the parasites were undergoing. CONCLUSIONS: The present results highlight the potential use of phenalenone derivatives against Leishmania species and further studies should be undertaken to establish them as novel leishmanicidal therapeutic agents.

LeishIF4E1 Deletion Affects the Promastigote Proteome, Morphology, and Infectivity.

Leishmania parasites cycle between sand-fly vectors and mammalian hosts, adapting to changing environmental conditions by driving a stage-specific program of gene expression, which is tightly regulated by translation processes. Leishmania encodes six eIF4E orthologs (LeishIF4Es) and five eIF4G candidates, forming different cap-binding complexes with potentially varying functions. Most LeishIF4E paralogs display temperature sensitivity in their cap-binding activity, except for LeishIF4E1, which maintains its cap-binding activity under all conditions. We used the CRISPR-Cas9 system to successfully generate a null mutant of LeishIF4E1 and examine how its elimination affected parasite physiology. Although the LeishIF4E1-/- null mutant was viable, its growth was impaired, in line with a reduction in global translation. As a result of the mutation, the null LeishIF4E1-/- mutant had a defective morphology, as the cells were round and unable to grow a normal flagellum. This was further emphasized when the LeishIF4E1-/- cells failed to develop the promastigote morphology once they shifted from conditions that generate axenic amastigotes (33°C, pH 5.5) back to neutral pH and 25°C, and they maintained their short flagellum and circular structure. Finally, the LeishIF4E1-/- null mutant displayed difficulty in infecting cultured macrophages. The morphological changes and reduced infectivity of the mutant may be related to differences in the proteomic profile of LeishIF4E1-/- cells from that of controls. All defects monitored in the LeishIF4E1-/- null mutant were reversed in the add-back strain, in which expression of LeishIF4E1 was reconstituted, establishing a strong link between the cellular defects and the absence of LeishIF4E1 expression.IMPORTANCELeishmania parasites are the causative agents of a broad spectrum of diseases. The parasites migrate between sand-fly vectors and mammalian hosts, adapting to changing environments by driving a regulated program of gene expression, with translation regulation playing a key role. The leishmanias encode six different paralogs of eIF4E, the cap-binding translation initiation factor. Since these vary in function, expression profile, and assemblage, it is assumed that each is assigned a specific role throughout the life cycle. Using the CRISPR-Cas9 system for Leishmania, we generated a null mutant of LeishIF4E1, eliminating both alleles. Although the mutant cells were viable, their morphology was altered and their ability to synthesize the flagellum was impaired. Elimination of LeishIF4E1 affected their protein expression profile and decreased their ability to infect cultured macrophages. Restoring LeishIF4E1 expression restored the affected features. This study highlights the importance of LeishIF4E1 in diverse cellular events during the life cycle of Leishmania.

Flavonoids induce cell death in Leishmania amazonensis: in vitro characterization by flow cytometry and Raman spectroscopy.

Leishmaniasis comprises a group of infectious diseases with worldwide distribution, of which both the visceral and cutaneous forms are caused by Leishmania parasites. In the absence of vaccines, efficacious chemotherapy remains the basis for leishmaniasis control. The available drugs are expensive and associated with several secondary adverse effects. Due to these limitations, the development of new antileishmanial compounds is imperative, and plants offer various perspectives in this regard. The present study evaluated the in vitro leishmanicidal activity of flavonoids isolated from Solanum paludosum Moric. and investigated the mechanisms of cell death induced by them. These compounds were evaluated in vitro for their antileishmanial activity against Leishmania amazonensis promastigotes and they showed prominent leishmanicidal activity. The EtOAc fraction, gossypetin 3,7,8,4'-tetra-O-methyl ether (1), and kaempferol 3,7-di-O-methyl ether (3) were selected to be used in an in vitro assay against L. amazonensis amastigotes and cell death assays. The flavonoids (1) and (3) presented significant activity against L. amazonensis amastigotes, exhibiting the IC50 values of 23.3 ± 4.5 µM, 34.0 ± 9.6 µM, and 10.5 ± 2.5 µM for the EtOAc fraction, (1), and (3), respectively, without toxic effects to the host cells. Moreover, (1) and (3) induced blocked cell cycle progression at the G1/S transition, ultimately leading to G1/G0 arrest. Flavonoid (3) also induced autophagy. Using Raman spectroscopy in conjunction with principal component analysis, the biochemical changes in the cellular components induced by flavonoids (1) and (3) were presented. The obtained results indicated that the mechanisms of action of (1) and (3) occurred through different routes. The results support that the flavonoids derived from S. paludosum can become lead molecules for the design of antileishmanial prototypes.

Quantification of Parasite Loads by Automated Microscopic Image Analysis.

High content analysis enables automated, robust, and unbiased evaluation of in vitro Leishmania infection. Here, we describe a protocol based on the infection of THP-1 macrophages with Leishmania promastigotes and the quantification of parasite load by high content analysis. The technique is capable of detecting and quantifying intracellular amastigotes, providing a multiparametric readout of the total number of cells, ratio of infected cells, total number of parasites, and number of parasites per infected cells. The technique can be used to quantitate infection of any Leishmania species in virtually all types of permissive host cells and can be applied to quantification of drug activity and studies of the Leishmania intracellular life cycle stage.

(De)glutamylation and cell death in Leishmania parasites.

Trypanosomatids are flagellated protozoan parasites that are very unusual in terms of cytoskeleton organization but also in terms of cell death. Most of the Trypanosomatid cytoskeleton consists of microtubules, forming different substructures including a subpellicular corset. Oddly, the actin network appears structurally and functionally different from other eukaryotic actins. And Trypanosomatids have an apoptotic phenotype under cell death conditions, but the pathways involved are devoid of key mammal proteins such as caspases or death receptors, and the triggers involved in apoptotic induction remain unknown. In this article, we have studied the role of the post-translational modifications, deglutamylation and polyglutamylation, in Leishmania. We have shown that Leishmania apoptosis was linked to polyglutamylation and hypothesized that the cell survival process autophagy was linked to deglutamylation. A balance seems to be established between polyglutamylation and deglutamylation, with imbalance inducing microtubule or other protein modifications characterizing either cell death if polyglutamylation was prioritized, or the cell survival process of autophagy if deglutamylation was prioritized. This emphasizes the role of post-translational modifications in cell biology, inducing cell death or cell survival of infectious agents.

In vitro leishmanicidal activity and theoretical insights into biological action of ruthenium(II) organometallic complexes containing anti-inflammatories.

Leishmaniasis, a neglected tropical disease caused by protozoans of the genus Leishmania, kills around 20-30 thousand people in Africa, Asia, and Latin America annually and, despite its potential lethality, it can be treated and eventually cured. However, the current treatments are limited owing to severe side effects and resistance development by some Leishmania. These factors make it urgent to develop new leishmanicidal drugs. In the present study, three ruthenium(II) organometallic complexes containing as ligands the commercially available anti-inflammatories diclofenac (dic), ibuprofen (ibu), and naproxen (nap) were synthesized, characterized, and subjected to in vitro leishmanicidal activity. The in vitro cytotoxicity assays against Leishmania (L.) amazonensis and Leishmania (L.) infantum promastigotes have shown that complexes [RuCl(dic)(η6-p-cymene)] (1) and [RuCl(nap)(η6-p-cymene)] (3) were active against both Leishmania species. Complex [RuCl(ibu)(η6-p-cymene)] (2) has exhibited no activity. The IC50 values for the two active complexes were respectively 7.42 and 23.55 µM, for L. (L.) amazonensis, and 8.57 and 42.25 µM, for L. (L.) infantum. Based on the toxicological results and computational analysis, we proposed a correlation between the complexes and their activity. Our results suggest both complexation to ruthenium(II) and ligands structure are key elements to leishmanicidal activity.

Development of an automated image analysis protocol for quantification of intracellular forms of Leishmania spp.

Leishmania parasites cause a set of neglected tropical diseases with considerable public health impact, the leishmaniases, which are often fatal if left untreated. Since current treatments for the leishmaniases exhibit high toxicity, low efficacy and prohibitive prices, many laboratories throughout the world are engaged in research for the discovery of novel chemotherapeutics. This entails the necessity of screening large numbers of compounds against the clinically relevant form of the parasite, the obligatory intracellular amastigote, a procedure that in many laboratories is still carried out by manual inspection. To overcome this well-known bottleneck in Leishmania drug development, several studies have recently attempted to automate this process. Here we implemented an image-based high content triage assay for Leishmania which has the added advantages of using primary macrophages instead of macrophage cell lines and of enabling identification of active compounds against parasite species developing both in small individual phagolysosomes (such as L. infantum) and in large communal vacuoles (such as L. amazonensis). The automated image analysis protocol is made available for IN Cell Analyzer systems, and, importantly, also for the open-source CellProfiler software, in this way extending its implementation to any laboratory involved in drug development as well as in other aspects of Leishmania research requiring analysis of in vitro infected macrophages.

Understanding Leishmania parasites through proteomics and implications for the clinic.

INTRODUCTION: Leishmania spp. are causative agents of leishmaniasis, a broad-spectrum neglected vector-borne disease. Genomic and transcriptional studies are not capable of solving intricate biological mysteries, leading to the emergence of proteomics, which can provide insights into the field of parasite biology and its interactions with the host. Areas covered: The combination of genomics and informatics with high throughput proteomics may improve our understanding of parasite biology and pathogenesis. This review analyses the roles of diverse proteomic technologies that facilitate our understanding of global protein profiles and definition of parasite development, survival, virulence and drug resistance mechanisms for disease intervention. Additionally, recent innovations in proteomics have provided insights concerning the drawbacks associated with conventional chemotherapeutic approaches and Leishmania biology, host-parasite interactions and the development of new therapeutic approaches. Expert commentary: With progressive breakthroughs in the foreseeable future, proteome profiles could provide target molecules for vaccine development and therapeutic intervention. Furthermore, proteomics, in combination with genomics and informatics, could facilitate the elimination of several diseases. Taken together, this review provides an outlook on developments in Leishmania proteomics and their clinical implications.

Novel antimony(iii) hydroxamic acid complexes as potential anti-leishmanial agents.

Reaction of benzohydroxamic acid (Bha), 2-pyridinehydroxamic acid (2-pyha), 2-amino-phenylhydroxamic acid (2-NH2-pha) and salicylhydroxamic acid (Sha) with SbCl3 in ethanol gave the corresponding novel hydroxamato Sb(iii) complexes, [Sb(Bha-1H)2Cl] 1, [SbCl2(2-pyha-1H)] 2, [Sb(2-NH2-pha-1H)(2-NH3-pha-1H)]Cl23 and [SbCl(Sha-1H)2] 4. In all cases the hydroxamic acids coordinate to the Sb centres in the typical bidentate hydroxamato (O,O') coordination mode, via the carbonyl oxygen and deprotonated hydroxyl group. Reaction of the histone deacetylase inhibitor (HDACi) suberoylanilidehydroxamic acid (SAHA) with Sb(OEt)3 gave the Sb(iii) hydroxamato/hydroximato complex, [Sb(SAHA-1H)(SAHA-2H)] 6. All test complexes significantly inhibited the promastigote proliferation of Leishmania amazonensis and L. chagasi and induced substantial changes in the general morphology of the parasites, including reduction in size and loss of flagellum, when compared to the untreated promastigotes. A dose-response approach using the test complexes showed a decreased in plasma membrane permeability and the mitochondrial dehydrogenase activities of the Leishmania species. [Sb(Bha-1H)2Cl] exhibited the best activity and was superior to the Sb HDACi complex 6. Though 1 exhibited noteworthy anti-leishmanial activity, the selectivity indexes determined suggest that [Sb(2-NH2-pha-1H)(2-NH3-pha-1H)]Cl23 is the test complex that merits further investigation as a potential anti-leishmanial agent.

Response of monoflagellate pullers to a shearing flow: A simulation study of microswimmer guidance.

Microscale swimming may be intuited to be dominated by background flows, sweeping away any untethered bodies with the prevalent flow direction. However, it has been observed that many microswimmers utilize ambient flows as guidance cues, in some cases resulting in net motion upstream, contrary to the dominant background fluid direction and our accompanying intuition. Thus the hydrodynamic response of small-scale motile organisms requires careful analysis of the complex interaction between swimmer and environment. Here we investigate the effects of a Newtonian shear flow on monoflagellated swimmers with specified body symmetry, representing, for instance, the Leishmania mexicana promastigote, a parasitic hydrodynamic puller that inhabits the microenvironment of a sandfly vector midgut and is the cause of a major and neglected human tropical disease. We observe that a lack of symmetry-breaking cellular geometry results in the periodic tumbling of swimmers in the bulk, with the rotations exhibiting a linear response to changes in shearing rate enabling analytic approximation. In order to draw comparisons with the better-studied pushers, we additionally consider virtual Leishmania promastigotes in a confined but typical geometry, that of a no-slip planar solid boundary, and note that in general stable guided taxis is not exhibited amongst the range of observed behaviors. However, a repulsive boundary gives rise to significant continued taxis in the presence of shearing flow, a phenomenon that may be of particular pertinence to the infective lifecycle stage of such swimmers subject to the assumption of a Newtonian medium. We finally propose a viable and general in vitro method of controlling microswimmer boundary accumulation using temporally evolving background shear flows, based on the analysis of phase-averaged dynamics and verified in silico.

Calcein+/PI- as an early apoptotic feature in Leishmania.

Although leishmaniases are responsible for high morbidity and mortality all over the world, no really satisfying treatment exists. Furthermore, the corresponding parasite Leishmania undergoes a very characteristic form of programmed cell death. Indeed, different stimuli can induce morphological and biochemical apoptotic-like features. However, the key proteins involved in mammal apoptosis, such as caspases and death receptors, are not encoded in the genome of this parasite. Currently, little is known about Leishmania apoptosis, notably owing to the lack of specific tools for programmed cell death analysis in these parasites. Furthermore, there is a need for a better understanding of Leishmania programmed cell death in order (i) to better understand the role of apoptosis in unicellular organisms, (ii) to better understand apoptosis in general through the study of an ancestral eukaryote, and (iii) to identify new therapeutic targets against leishmaniases. To advance understanding of apoptosis in Leishmania, in this study we developed a new tool based on the quantification of calcein and propidium iodide by flow cytometry. This double labeling can be employed to distinguish early apoptosis, late apoptosis and necrosis in Leishmania live cells with a very simple and rapid assay. This paper should, therefore, be of interest for people working on Leishmania and related parasites.