Ring Stage Research Articles

Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax.

Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species.

Characterization of native PfABCG protein in Plasmodium falciparum

The Plasmodium falciparum genome encodes 16 members of ABC proteins, with one member of the ABCG subfamily (PfABCG). Analysis of PfABCG amino acid sequence shows equal sequence identity to hsABCG1 and G2. Using N-terminal directed antibody against a recombinant fragment of PfABCG, we show that PfABCG migrates with an apparent molecular mass of 65KDa polypeptide on SDS-PAGE. PfABCG is expressed in all four stages of the parasite erythrocytic life cycle, with lower and higher expression in ring and late trophozoite stages, respectively. The protein localizes to the plasma membrane and a novel spherical structure beneath the cell membrane. Similar localization is also observed in gametocytes where PfABCG is highly expressed. Analysis of PfABCG genomic sequences for polymorphisms and changes in protein expression between different strains of P. falciparum revealed identical nucleotide sequence among the different strains, but variable protein expression. PfABCG expression is least in HB3 chloroquine sensitive strain, while higher expression levels are seen in other chloroquine-sensitive and -resistant strains, with highest levels of expression in 7G8. The differential expression of PfABCG in three chloroquine-sensitive strains (e.g., 3D7, HB3 and D10) predicts the sensitivity of the different strains to ketotifen, an anti-histaminic drug, whereby low expression is associated with decreased sensitivity to ketotifen. Taken together, the results in this report provide the first description of native PfABCG expression and subcellular localization in asexual stages of the parasite and its localization in gametocytes. It remains to be determined if PfABCG is functionally equivalent to mammalian ABCG1, ABCG2 or both.

Potent Ex Vivo Activity of Naphthoquine and Methylene Blue against Drug-Resistant Clinical Isolates of Plasmodium falciparum and Plasmodium vivax.

The 4-aminoquinoline naphthoquine (NQ) and the thiazine dye methylene blue (MB) have potent in vitro efficacies against Plasmodium falciparum, but susceptibility data for P. vivax are limited. The species- and stage-specific ex vivo activities of NQ and MB were assessed using a modified schizont maturation assay on clinical field isolates from Papua, Indonesia, where multidrug-resistant P. falciparum and P. vivax are prevalent. Both compounds were highly active against P. falciparum (median [range] 50% inhibitory concentration [IC50]: NQ, 8.0 nM [2.6 to 71.8 nM]; and MB, 1.6 nM [0.2 to 7.0 nM]) and P. vivax (NQ, 7.8 nM [1.5 to 34.2 nM]; and MB, 1.2 nM [0.4 to 4.3 nM]). Stage-specific drug susceptibility assays revealed significantly greater IC50s in parasites exposed at the trophozoite stage than at the ring stage for NQ in P. falciparum (26.5 versus 5.1 nM, P = 0.021) and P. vivax (341.6 versus 6.5 nM, P = 0.021) and for MB in P. vivax (10.1 versus 1.6 nM, P = 0.010). The excellent ex vivo activities of NQ and MB against both P. falciparum and P. vivax highlight their potential utility for the treatment of multidrug-resistant malaria in areas where both species are endemic.

Contrasting ex vivo efficacies of "reversed chloroquine" compounds in chloroquine-resistant Plasmodium falciparum and P. vivax isolates.

Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance ex vivo. Between March to October 2011 and January to September 2013, two "reversed chloroquine" (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of Plasmodium falciparum (n = 41) and Plasmodium vivax (n = 45) in Papua, Indonesia, using a modified ex vivo schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant P. falciparum and P. vivax field isolates. For P. falciparum, the median 50% inhibitory concentrations (IC50s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (P < 0.001 and P = 0.036, respectively). The corresponding values for P. vivax were 19.0, 60.0, and 60.9 nM (P < 0.001 and P = 0.018, respectively). There was a significant correlation between IC50s of CQ and PL69 (Spearman's rank correlation coefficient [r s] = 0.727, P < 0.001) and PL106 (rs = 0.830, P < 0.001) in P. vivax but not in P. falciparum. Both RCQs were equally active against the ring and trophozoite stages of P. falciparum, but in P. vivax, PL69 and PL106 showed less potent activity against trophozoite stages (median IC50s, 130.2 and 172.5 nM) compared to ring stages (median IC50s, 17.6 and 91.3 nM). RCQ compounds have enhanced ex vivo activity against CQ-resistant clinical isolates of P. falciparum and P. vivax, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two Plasmodium species.

Enhancing malaria diagnosis through microfluidic cell enrichment and magnetic resonance relaxometry detection.

Despite significant advancements over the years, there remains an urgent need for low cost diagnostic approaches that allow for rapid, reliable and sensitive detection of malaria parasites in clinical samples. Our previous work has shown that magnetic resonance relaxometry (MRR) is a potentially highly sensitive tool for malaria diagnosis. A key challenge for making MRR based malaria diagnostics suitable for clinical testing is the fact that MRR baseline fluctuation exists between individuals, making it difficult to detect low level parasitemia. To overcome this problem, it is important to establish the MRR baseline of each individual while having the ability to reliably determine any changes that are caused by the infection of malaria parasite. Here we show that an approach that combines the use of microfluidic cell enrichment with a saponin lysis before MRR detection can overcome these challenges and provide the basis for a highly sensitive and reliable diagnostic approach of malaria parasites. Importantly, as little as 0.0005% of ring stage parasites can be detected reliably, making this ideally suited for the detection of malaria parasites in peripheral blood obtained from patients. The approaches used here are envisaged to provide a new malaria diagnosis solution in the near future.

Development of cultured Plasmodium falciparum blood-stage malaria cell banks for early phase in vivo clinical trial assessment of anti-malaria drugs and vaccines.

BackgroundThe ability to undertake controlled human malaria infection (CHMI) studies for preliminary evaluation of malaria vaccine candidates and anti-malaria drug efficacy has been limited by the need for access to sporozoite infected mosquitoes, aseptic, purified, cryopreserved sporozoites or blood-stage malaria parasites derived ex vivo from malaria infected individuals. Three different strategies are described for the manufacture of clinical grade cultured malaria cell banks suitable for use in CHMI studies.MethodsGood Manufacturing Practices (GMP)-grade Plasmodium falciparum NF54, clinically isolated 3D7, and research-grade P. falciparum 7G8 blood-stage malaria parasites were cultured separately in GMP-compliant facilities using screened blood components and then cryopreserved to produce three P. falciparum blood-stage malaria cell banks. These cell banks were evaluated according to specific criteria (parasitaemia, identity, viability, sterility, presence of endotoxin, presence of mycoplasma or other viral agents and in vitro anti-malarial drug sensitivity of the cell bank malaria parasites) to ensure they met the criteria to permit product release according to GMP requirements.ResultsThe P. falciparum NF54, 3D7 and 7G8 cell banks consisted of >78% ring stage parasites with a ring stage parasitaemia of >1.4%. Parasites were viable in vitro following thawing. The cell banks were free from contamination with bacteria, mycoplasma and a broad panel of viruses. The P. falciparum NF54, 3D7 and 7G8 parasites exhibited differential anti-malarial drug susceptibilities. The P. falciparum NF54 and 3D7 parasites were susceptible to all anti-malaria compounds tested, whereas the P. falciparum 7G8 parasites were resistant/had decreased susceptibility to four compounds. Following testing, all defined release criteria were met and the P. falciparum cell banks were deemed suitable for release. Ethical approval has been obtained for administration to human volunteers.ConclusionsThe production of cultured P. falciparum blood-stage malaria cell banks represents a suitable approach for the generation of material suitable for CHMI studies. A key feature of this culture-based approach is the ability to take research-grade material through to a product suitable for administration in clinical trials.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0663-x) contains supplementary material, which is available to authorized users.

Discovery of new thienopyrimidinone derivatives displaying antimalarial properties toward both erythrocytic and hepatic stages of Plasmodium

A preliminary in vitro screening of compounds belonging to various chemical families from our library revealed the thieno[3,2-d]pyrimidin-4(3H)-one scaffold displayed a promising profile against Plasmodium falciparum. Then, 120 new derivatives were synthesized and evaluated in vitro; compared to drug references, 40 showed good activity toward chloroquine sensitive (IC50 35–344 nM) and resistant (IC50 45–800 nM) P. falciparum strains. They were neither cytotoxic (CC50 15–50 μM) toward HepG2 and CHO cells, nor mutagenic. Structure–activity relationships were defined. The lead-compound also appeared active against the Plasmodium liver stages (Plasmodium yoelii IC50 = 35 nM) and a preliminary in vivo evaluation indicated the in vitro activity was preserved (45% reduction in parasitemia compared to untreated infected mice). A mechanistic study demonstrated these molecules do not involve any of the pathways described for commercial drugs and exert a specific activity on the ring and trophozoite stages.

Inhibition of protein synthesis and malaria parasite development by drug targeting of methionyl-tRNA synthetases.

Aminoacyl-tRNA synthetases (aaRSs) are housekeeping enzymes that couple cognate tRNAs with amino acids to transmit genomic information for protein translation. The Plasmodium falciparum nuclear genome encodes two P. falciparum methionyl-tRNA synthetases (PfMRS), termed PfMRS(cyt) and PfMRS(api). Phylogenetic analyses revealed that the two proteins are of primitive origin and are related to heterokonts (PfMRS(cyt)) or proteobacteria/primitive bacteria (PfMRS(api)). We show that PfMRS(cyt) localizes in parasite cytoplasm, while PfMRS(api) localizes to apicoplasts in asexual stages of malaria parasites. Two known bacterial MRS inhibitors, REP3123 and REP8839, hampered Plasmodium growth very effectively in the early and late stages of parasite development. Small-molecule drug-like libraries were screened against modeled PfMRS structures, and several "hit" compounds showed significant effects on parasite growth. We then tested the effects of the hit compounds on protein translation by labeling nascent proteins with (35)S-labeled cysteine and methionine. Three of the tested compounds reduced protein synthesis and also blocked parasite growth progression from the ring stage to the trophozoite stage. Drug docking studies suggested distinct modes of binding for the three compounds, compared with the enzyme product methionyl adenylate. Therefore, this study provides new targets (PfMRSs) and hit compounds that can be explored for development as antimalarial drugs.

Haemoglobin degradation underpins the sensitivity of early ring stage Plasmodium falciparum to artemisinins.

ABSTRACTCurrent first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance.

Measuring Plasmodium falciparum Erythrocyte Invasion Phenotypes Using Flow Cytometry.

Having the ability to rapidly, accurately, and robustly measure Plasmodium falciparum merozoite invasion is a critical component in effective assessment of a blood stage vaccine's mechanism of action. Being able to measure invasion of erythrocytes accurately, objectively and in a high throughput fashion is of critical importance. Here, we describe a simple and robust flow cytometry method that allows for the measurement of the key invasion parameters of parasite multiplication rate and erythrocyte selectivity-both important determinants of disease severity-from the schizont to the ring stage of the parasite's life-cycle, thus separating invasion from growth of the parasite. Importantly, this method is able to accurately detect low levels of parasitemia and heterogeneity within the population that can be missed by enzymatic methods. Lastly, this method has been successfully adapted and employed in field based research settings for parasitemia measurements in vivo, ex vivo, and in vitro and to measure invasion inhibition by antibodies and the use of alternative pathways for invasion.

Transcriptional profiling defines dynamics of parasite tissue sequestration during malaria infection.

BackgroundDuring intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes - sexual stage precursor cells - likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the bloodstream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood.ResultsWe generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post-invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and approximately 30 hours post-invasion and mature gametocytes after around 7 days post-invasion.ConclusionsThis study provides a bioinformatics resource for the functional elucidation of parasite life cycle dynamics and specifically demonstrates the presence of the gametocyte ring stages in circulation, adding significantly to our understanding of the dynamics of gametocyte sequestration in vivo.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-015-0133-7) contains supplementary material, which is available to authorized users.

Senicapoc, a Gardos Channel Inhibitor Developed to Treat Sickle Cell Disease, Exhibits Antimalarial Activity

▪ BACKGROUNDThe inhibitor of the erythrocyte Gardos channel (KCNN-4, IK-1), senicapoc, was developed for treatment of sickle cell disease. Activation of the Gardos channel constitutes a major potassium leak pathway contributing to cellular dehydration. Administration of senicapoc to patients with sickle cell disease was well tolerated and reduced hemolysis through attenuation of sickle red cell dehydration, but failed to reduce the frequency of vaso-occlusive pain crises (Ataga KI et al. Br J Haematol 2011;153:92-104). Red cell volume regulation is critical both to the pathology of sickle cell disease and to the growth of Plasmodium, the parasites that cause malaria, since Plasmodium depends upon ion flow across the host membrane for growth (Glushakova S et al. Curr Biol 2010; 20:1117-1121). We tested the hypothesis that senicapoc-induced blockade of the Gardos channel inhibits intraerythrocytic growth and development of malarial parasites. METHODSBlood type O+ erythrocytes were infected with P. falciparum strains 3D7, W2mef, and 7G8, and P. knowlesi strains H-1 and YH-1. Parasites were cultured in the presence of senicapoc and in parallel, with drugs with known antimalarial properties (e.g. mefloquine, chloroquine, clotrimazole, and dihydroartemisinin). Parasites were also cultured in the presence of each of eight senicapoc analogs with variable inhibitory potency for the Gardos channel. To define interactions between these drugs, parasites were cultured in fixed-ratio combinations of senicapoc and senicapoc analogs using the fixed-ratio isobologram method. Parasite growth and maturation were determined by microscopy and flow cytometry. The effect of senicapoc in vivo was determined using wild-type C57Bl/6 mice along with IK-1+/+ and IK-1-/- pups on a C57Bl/6 background. Mice were infected with the murine parasite, P. yoelii and treated with senicapoc or vehicle control (methylcellulose 0.5%). RESULTSWe observed that senicapoc blocked the intraerythrocyte growth of P. falciparum 3D7 with an IC50 value of 7 uM and P. knowlesi H1 with an IC50 value of 18 uM. Senicapoc inhibited growth leading to death of intracellular parasites at and beyond the late trophozoite stage, demonstrating minimal effect on ring stage parasites, and an intermediate effect on mature schizonts. Analogs with IC50 values for inhibition of parasite growth similar to senicapoc were potent inhibitors of the Gardos channel. These analogs also shared common elements of the molecular structure. Combined treatment with senicapoc and analogs with potent inhibition of the Gardos channel demonstrated an additive effect on inhibition of parasite growth. Both additive and synergistic interactions were demonstrated when parasites were cultured with senicapoc and analogs without potent inhibition of the Gardos channel. In the murine model, C57Bl/6 mice treated with senicapoc exhibited suppression in parasite growth, confirming the efficacy of senicapoc against Plasmodia in vivo. Parasite growth was suppressed in IK+/+ mice treated with senicapoc, though the effect was not sustained through the period of treatment. Surprisingly, parasite growth was also suppressed in IK-/-mice treated with senicapoc, though the response was delayed. The kinetics of parasite growth differed between mice with and without the Gardos channel. CONCLUSIONSSenicapoc demonstrates antimalarial activity against P. falciparum and P. knowlesiin vitro and in vivo against P. yoelii in mice. Our in vivo studies suggest a mechanism for inhibition of parasite growth in mice that includes both Gardos-dependent and independent components. The excellent safety profile of senicapoc and its long half-life in humans demonstrated in clinical trials suggest its possible utility in antimalarial development either as a lead compound or in combination with other antimalarials. DisclosuresNo relevant conflicts of interest to declare.

Genome wide in silico analysis of Plasmodium falciparum phosphatome.

BackgroundEukaryotic cellular machineries are intricately regulated by several molecular mechanisms involving transcriptional control, post-translational control and post-translational modifications of proteins (PTMs). Reversible protein phosphorylation/dephosphorylation process, which involves kinases as well as phosphatases, represents an important regulatory mechanism for diverse pathways and systems in all organisms including human malaria parasite, Plasmodium falciparum. Earlier analysis on P. falciparum protein-phosphatome revealed presence of 34 phosphatases in Plasmodium genome. Recently, we re-analysed P. falciparum phosphatome aimed at identifying parasite specific phosphatases.ResultsPlasmodium database (PlasmoDB 9.2) search, combined with PFAM and CDD searches, revealed 67 candidate phosphatases in P. falciparum. While this number is far less than the number of phosphatases present in Homo sapiens, it is almost the same as in other Plasmodium species. These Plasmodium phosphatase proteins were classified into 13 super families based on NCBI CDD search. Analysis of proteins expression profiles of the 67 phosphatases revealed that 44 phosphatases are expressed in both schizont as well as gametocytes stages. Fourteen phosphatases are common in schizont, ring and trophozoite stages, four phosphatases are restricted to gametocytes, whereas another three restricted to schizont stage. The phylogenetic trees for each of the known phosphatase super families reveal a considerable phylogenetic closeness amongst apicomplexan organisms and a considerable phylogenetic distance with other eukaryotic model organisms included in the study. The GO assignments and predicted interaction partners of the parasite phosphatases indicate its important role in diverse cellular processes.ConclusionIn the study presented here, we reviewed the P. falciparum phosphatome to show presence of 67 candidate phosphatases in P. falciparum genomes/proteomes. Intriguingly, amongst these phosphatases, we could identify six Plasmodium specific phosphatases and 33 putative phosphatases that do not have human orthologs, thereby suggesting that these phosphatases have the potential to be explored as novel antimalarial drug targets.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1024) contains supplementary material, which is available to authorized users.

High-throughput tri-colour flow cytometry technique to assess Plasmodium falciparum parasitaemia in bioassays.

BackgroundUnbiased flow cytometry-based methods have become the technique of choice in many laboratories for high-throughput, accurate assessments of malaria parasites in bioassays. A method to quantify live parasites based on mitotracker red CMXRos was recently described but consistent distinction of early ring stages of Plasmodium falciparum from uninfected red blood cells (uRBC) remains a challenge.MethodsHere, a high-throughput, three-parameter (tri-colour) flow cytometry technique based on mitotracker red dye, the nucleic acid dye coriphosphine O (CPO) and the leucocyte marker CD45 for enumerating live parasites in bioassays was developed. The technique was applied to estimate the specific growth inhibition index (SGI) in the antibody-dependent cellular inhibition (ADCI) assay and compared to parasite quantification by microscopy and mitotracker red staining. The Bland-Altman analysis was used to compare biases between SGI estimated by the tri-colour staining technique, mitotracker red and by microscopy.ResultsCPO allowed a better separation between early rings and uRBCs compared to mitotracker red resulting in a more accurate estimate of total parasitaemia. The tri-colour technique is rapid, cost effective and robust with comparable sensitivity to microscopy and capable of discriminating between live and dead and/or compromised parasites. Staining for CD45 improved parasitaemia estimates in ADCI assay since high numbers of leucocytes interfered with the accurate identification of parasitized RBC. The least bias (-1.60) in SGI was observed between the tri-colour and microscopy.ConclusionAn improved methodology for high-throughput assessment of P. falciparum parasitaemia under culture conditions that could be useful in different bioassays, including ADCI and growth inhibition assays has been developed.

Assessment of the induction of dormant ring stages in Plasmodium falciparum parasites by artemisone and artemisone entrapped in Pheroid vesicles in vitro.

The in vitro antimalarial activities of artemisone and artemisone entrapped in Pheroid vesicles were compared, as was their ability to induce dormancy in Plasmodium falciparum. There was no increase in the activity of artemisone entrapped in Pheroid vesicles against multidrug-resistant P. falciparum lines. Artemisone induced the formation of dormant ring stages similar to dihydroartemisinin. Thus, the Pheroid delivery system neither improved the activity of artemisone nor prevented the induction of dormant rings.

Profiling of the embryonic Atlantic halibut (Hippoglossus hippoglossus L.) transcriptome reveals maternal transcripts as potential markers of embryo quality.

BackgroundCommercial Atlantic halibut (Hippoglossus hippoglossus) farming is restricted by variable oocyte quality, slow growth, and early maturation of male fish. Maternally transferred components regulate early developmental processes; therefore, they have an effect on the future viability of the embryo. Using a newly developed Agilent 10 k custom-made oligonucleotide array, we profiled components of the transcriptome involved in immune defence as well as germline and muscle development during early developmental stages: 8-cell embryos (8CS), germ ring stage (GR), 10-somite stage (10SS), and hatched embryos (HT). In addition, we identified differentially expressed transcripts in low (≤9 ± 3% hatching) and high (≥86 ± 3°% hatching) quality eggs at 8CS to identify potential maternal markers for embryo quality.ResultsOut of 2066 differentially expressed transcripts, 160 were identified as maternal transcripts being specifically expressed at 8CS only. Twenty transcripts were differentially expressed in 8-cell embryos between low and high quality egg groups. Several immune-related transcripts were identified as promising molecular markers of hatching success including interferon regulatory factor 7 and mhc class 2A chain. Differential expression was positively validated with quantitative real-time PCR.ConclusionsWe have demonstrated maternal transfer of innate and adaptive immune system transcripts into Atlantic halibut embryos and their relation with future embryo developmental potential. We identified several transcripts as potential molecular markers of embryo quality. The developed microarray represents a useful resource for improving the commercial production of Atlantic halibut.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-829) contains supplementary material, which is available to authorized users.

Label-free microfluidic enrichment of ring-stage Plasmodium falciparum-infected red blood cells using non-inertial hydrodynamic lift.

BackgroundUnderstanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated.MethodsA simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker.ResultsThe possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of ~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated.ConclusionThe method allows for the enrichment of red blood cells early after the invasion by P. falciparum parasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic- or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.

Characterization of stress–strain behaviour of red blood cells (RBCs), part II: response of malaria-infected RBCs

Malaria is an infectious disease induced by a parasite named Plasmodium and annually affects two to three million people. The most serious form of malaria for humans is caused by Plasmodium falciparum, and the progression of P. falciparum is closely linked to the deformation characteristics of red blood cells (RBCs). Conventional isotropic material models have been used to represent the RBC behaviour. In this paper, an inverse analysis approach, Self-Learning Simulation (SelfSim), is applied to extract the RBC material behaviour from complementary boundary force and displacement measurements of healthy, exposed, ring, trophozoit and schizont stages of RBC obtained by optical tweezers method. SelfSim reveals that the nonlinear deformation characteristics of healthy RBC are lost with P. falciparum parasite development. The deformability and anisotropic stress–strain behaviour inferred from healthy RBC decrease for mature stages of malaria-infected RBCs. SelfSim provides new insights into the stress–strain behaviour maps of malaria progression for disease diagnosis.

In vitro antiplasmodial activity of cepharanthine.

BackgroundNew classes of anti-malarial drugs are needed to control the alarming Plasmodium falciparum resistance toward current anti-malarial therapy. The ethnopharmacological approach allows the discovery of original chemical structures from the vegetable biodiversity. Previous studies led to the selection of a bisbenzylisoquinoline, called cepharanthine and isolated from a Cambodian plant: Stephania rotunda. Cepharanthine could exert a mechanism of action different from commonly used drugs. Potential plasmodial targets are reported here.MethodsTo study the mechanism of action of cepharanthine, a combined approach using phenotypic and transcriptomic techniques was undertaken.ResultsCepharanthine blocked P. falciparum development in ring stage. On a culture of synchronized ring stage, the comparisons of expression profiles showed that the samples treated with 5 μM of cepharanthine (IC90) were significantly closer to the initial controls than to the final ones. After a two-way ANOVA (p-value < 0.05) on the microarray results, 1,141 probes among 9,722 presented a significant differential expression.A gene ontology analysis showed that the Maurer’s clefts seem particularly down-regulated by cepharanthine. The analysis of metabolic pathways showed an impact on cell-cell interactions (cytoadherence and rosetting), glycolysis and isoprenoid pathways. Organellar functions, more particularly constituted by apicoplast and mitochondrion, are targeted too.ConclusionThe blockage at the ring stage by cepharanthine is described for the first time. Transcriptomic approach confirmed that cepharanthine might have a potential innovative antiplasmodial mechanism of action. Thus, cepharanthine might play an ongoing role in the progress on anti-malarial drug discovery efforts.Electronic supplementary materialThe online version of this article (doi:10.1186/1475-2875-13-327) contains supplementary material, which is available to authorized users.

Maturation of Plasmodium falciparum in multiply infected erythrocytes and the potential role in malaria pathogenesis.

Erythrocytes containing two or more parasites, referred to here as multiply infected erythrocytes (MIEs), are common in the blood of humans infected by Plasmodium falciparum. It is necessary to study these cells closely because the excess numbers of parasites they contain suggest that they could be overloaded with virulence factors. Here, microscopic examinations of blood smears from patients showed that up to seven merozoites can successfully invade an erythrocyte and mature to ring stage. However, in vitro culture showed that only up to three parasites can mature to late schizont stage. These observations were made by culturing the parasites in erythrocytes containing hemoglobin AA (HbAA), HbAS, and HbSS. Biochemical analysis of saponin-concentrated culture suggests that more hemozoin is produced in a MIE than in a singly infected erythrocyte (SIE). Studies have shown that ingestion of excessive hemozoin destroys monocytes and neutrophils, which could impair the immune system. Cultured parasites were also examined by transmission electron microscopy, and it was found that the quantity of knobs was dramatically increased on the membranes of erythrocytes containing multiple schizonts, compared to those containing only one schizont. Knobs contain, among other things, P. falciparum erythrocyte membrane protein 1 (PfEMP1) complex which mediates sequestration and promotes severe malaria. These findings suggest that P. falciparum increases its virulence by producing MIEs. On sexual life cycle of the parasite, microphotographs are presented in this report showing, for the first time, that two gametocytes can develop in one erythrocyte; they are referred to here as twin gametocytes. It is not known whether they can infect mosquitoes.