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Purpose: To demonstrate the validity and reliability of a smartphone app to measure ROM after stroke.Materials and methods: Twenty-one stroke survivors with a diagnosis of stroke that affected the ...

OBJECTIVES Indolone-N-oxides are characterized by the presence of a highly reactive pharmacophore, the nitrone moiety (C=N(+)-O(-)), which undergoes oxidation-reduction reactions. The aims of the present study were to: (i) evaluate the in vitro activity of the parent compound, designated as compound 1, against 34 fresh clinical isolates of Plasmodium falciparum; (ii) compare the activity of compound 1 with that of chloroquine and dihydroartemisinin to assess the potential for cross-resistance; (iii) investigate drug interactions of indolone-N-oxides with standard antimalarials; and (iv) determine the stage-dependent activity of indolone-N-oxides. METHODS In vitro antimalarial activity was evaluated against clinical isolates collected from Cameroonian patients by the [(3)H]hypoxanthine incorporation assay. In vitro interactions between compound 1 or another analogue, compound 4, and established antimalarial drugs were assessed by the fixed ratio method. Stage specificity was evaluated by light microscopy using highly synchronized P. falciparum cultures. RESULTS The geometric mean 50% inhibitory concentration (IC(50)) of compound 1 was 48.6 nM. Its activity did not differ between the chloroquine-susceptible and the chloroquine-resistant isolates. There was no correlation between chloroquine and compound 1 responses (r = 0.015; P > 0.05), but the in vitro responses of compound 1 and dihydroartemisinin were significantly and positively correlated (r = 0.444; P

A series of 66 new indolone-N-oxide derivatives was synthesized with three different methods. Compounds were evaluated for in vitro activity against CQ-sensitive (3D7), CQ-resistant (FcB1), and CQ and pyrimethamine cross-resistant (K1) strains of Plasmodium falciparum (P.f.), as well as for cytotoxic concentration (CC(50)) on MCF7 and KB human tumor cell lines. Compound 26 (5-methoxy-indolone-N-oxide analogue) had the most potent antiplasmodial activity in vitro (3 nM on FcB1 and = 1.7 nM on 3D7) with a very satisfactory selectivity index (CC(50) MCF7/IC(50) FcB1: 14623; CC(50) KB/IC(50) 3D7: 198823). In in vivo experiments, compound 1 (dioxymethylene derivatives of the indolone-N-oxide) showed the best antiplasmodial activity against Plasmodium berghei, 62% inhibition of the parasitaemia at 30 mg/kg/day.

The main aim of the evaluation was to identify the strengths and weaknesses of the service in order to contribute to its further development and sustainability. The study was carried out in partnership between Lincolnshire Adult Social Care and Lincolnshire Primary Care Trust. A secondary aim was to determine the impact of the service on the quality of life and independence of service users using appropriate scales. These measurements were performed in parallel with an audit of the performance and cost effectiveness of the service. Service users, carers and staff all contributed to the evaluation. Initial results have indicated a positive response to the equipment, with a reduction in anxiety and increased independence for both the service user and carer.

The Plasmodium falciparum cGMP-dependent protein kinase (PfPKG) is a key regulator across the malaria parasite life cycle. Little is known about PfPKG’s activation mechanism. Here we report that the carboxyl cyclic nucleotide binding domain functions as a “gatekeeper” for activation by providing the highest cGMP affinity and selectivity. To understand the mechanism, we have solved its crystal structures with and without cGMP at 2.0 and 1.9 Å, respectively. These structures revealed a PfPKG-specific capping triad that forms upon cGMP binding, and disrupting the triad reduces kinase activity by 90%. Furthermore, mutating these residues in the parasite prevents blood stage merozoite egress, confirming the essential nature of the triad in the parasite. We propose a mechanism of activation where cGMP binding allosterically triggers the conformational change at the αC-helix, which bridges the regulatory and catalytic domains, causing the capping triad to form and stabilize the active conformation., Author Summary Malaria causes up to a million fatalities per year worldwide. Most of these deaths are caused by Plasmodium falciparum, which has a complex life cycle in both humans and mosquitoes. One key regulator of this process is P. falciparum cGMP-dependent protein kinase (PfPKG), the main effector of the cGMP-signaling pathway. Specifically blocking this kinase stops both replication and transmission of the parasites, suggesting that PfPKG is a promising drug target. Here we identified the carboxyl cGMP-binding domain of PfPKG serving as a gatekeeper for activation of the entire kinase by having the highest affinity and selectivity for cGMP. High-resolution crystal structures with and without cGMP allowed us to identify a novel cGMP capping triad that dynamically forms upon binding cGMP and stabilizes the activated conformation. Mutation of the capping triad forming residues not only reduces its kinase activity, but also prevents blood stage merozoite egress, demonstrating its crucial role in PfPKG activation.

The emergence of drug-resistant parasites is a serious threat faced by malaria control programs. Understanding the genetic basis of resistance is critical to the success of treatment and intervention strategies. A novel locus associated with antimalarial resistance, ap2-mu (encoding the mu chain of the adaptor protein 2 [AP2] complex), was recently identified in studies on the rodent malaria parasite Plasmodium chabaudi (pcap2-mu). Furthermore, analysis in Kenyan malaria patients of polymorphisms in the Plasmodium falciparum ap2-mu homologue, pfap2-mu, found evidence that differences in the amino acid encoded by codon 160 are associated with enhanced parasite survival in vivo following combination treatments which included artemisinin derivatives. Here, we characterize the role of pfap2-mu in mediating the in vitro antimalarial drug response of P. falciparum by generating transgenic parasites constitutively expressing codon 160 encoding either the wild-type Ser (Ser160) or the Asn mutant (160Asn) form of pfap2-mu. Transgenic parasites carrying the pfap2-mu 160Asn allele were significantly less sensitive to dihydroartemisinin using a standard 48-h in vitro test, providing direct evidence of an altered parasite response to artemisinin. Our data also provide evidence that pfap2-mu variants can modulate parasite sensitivity to quinine. No evidence was found that pfap2-mu variants contribute to the slow-clearance phenotype exhibited by P. falciparum in Cambodian patients treated with artesunate monotherapy. These findings provide compelling evidence that pfap2-mu can modulate P. falciparum responses to multiple drugs. We propose that this gene should be evaluated further as a potential molecular marker of antimalarial resistance.

Chemical genetics and a global comparative analysis of phosphorylation and phospholipids in vivo shows that PKG is the upstream regulator that induces calcium signals that enables Plasmodium to progress through its complex life cycle., Many critical events in the Plasmodium life cycle rely on the controlled release of Ca2+ from intracellular stores to activate stage-specific Ca2+-dependent protein kinases. Using the motility of Plasmodium berghei ookinetes as a signalling paradigm, we show that the cyclic guanosine monophosphate (cGMP)-dependent protein kinase, PKG, maintains the elevated level of cytosolic Ca2+ required for gliding motility. We find that the same PKG-dependent pathway operates upstream of the Ca2+ signals that mediate activation of P. berghei gametocytes in the mosquito and egress of Plasmodium falciparum merozoites from infected human erythrocytes. Perturbations of PKG signalling in gliding ookinetes have a marked impact on the phosphoproteome, with a significant enrichment of in vivo regulated sites in multiple pathways including vesicular trafficking and phosphoinositide metabolism. A global analysis of cellular phospholipids demonstrates that in gliding ookinetes PKG controls phosphoinositide biosynthesis, possibly through the subcellular localisation or activity of lipid kinases. Similarly, phosphoinositide metabolism links PKG to egress of P. falciparum merozoites, where inhibition of PKG blocks hydrolysis of phosphatidylinostitol (4,5)-bisphosphate. In the face of an increasing complexity of signalling through multiple Ca2+ effectors, PKG emerges as a unifying factor to control multiple cellular Ca2+ signals essential for malaria parasite development and transmission., Author Summary Malaria, caused by Plasmodium spp. parasites, is a profound human health problem. Plasmodium parasites progress through a complex life cycle as they move between infected humans and blood-feeding mosquitoes. We know that tight regulation of calcium ion levels within the cytosol of the parasite is critical to control multiple signalling events in their life cycle. However, how these calcium levels are controlled remains a mystery. Here, we show that a single protein kinase, the cGMP-dependent protein kinase G (PKG), controls the calcium signals that are critical at three different points of the life cycle: (1) for the exit of the merozoite form of the parasite from human erythrocytes (red blood cells), (2) for the cellular activation that happens when Plasmodium sexual transmission stages are ingested by a blood-feeding mosquito, and (3) for the productive gliding of the ookinete, which is the parasite stage that invades the mosquito midgut. We provide initial evidence that the universal role of PKG relies on the production of lipid precursors which then give rise to inositol (1,4,5)-trisphosphate (IP3), a messenger molecule that serves as a signal for the release of calcium from stores within the parasite. This signalling pathway provides a potential target to block both malaria development in the human host and transmission to the mosquito vector.

OBJECTIVES: Curcumin is a natural plant product with antimalarial activity and immunomodulatory properties. In this study we aimed to investigate its effects on CD36 expression and CD36-mediated Plasmodium falciparum phagocytosis as well as the role played by reactive oxygen species (ROS) and the peroxisome proliferator-activated receptor ? retinoid X receptor (PPAR?-RXR) in these processes. METHODS: In vitro antimalarial activity was evaluated by the [(3)H]hypoxanthine assay. ROS production and surface CD36 in human monocyte/macrophages were measured by flow cytometry. PPAR? and CD36 mRNA expression was determined by the QuantiGene Plex(®) assay and RT-qPCR. Nuclear PPAR? activation was analysed by a DNA-binding ELISA while nuclear erythroid-related factor 2 (Nrf2) expression was analysed by western blotting. P. falciparum phagocytosis was assessed by light microscopy. RESULTS: Curcumin's antimalarial activity was confirmed and did not differ between drug-susceptible and -resistant P. falciparum strains. Curcumin increased monocyte ROS production and expression of PPAR? and CD36 at the mRNA and protein levels. Although PPAR? activation was blocked by the PPAR? antagonist GW9662, CD36 expression and CD36-mediated P. falciparum phagocytosis were only inhibited by N-acetylcysteine (NAC), suggesting a PPAR?-independent CD36 expression pathway. We then identified seven putative Nrf2 antioxidant response elements on the CD36 gene promoter and showed that NAC inhibited curcumin-induced Nrf2 protein expression. CONCLUSIONS: CD36 expression and CD36-mediated P. falciparum phagocytosis by curcumin are dependent on ROS production and probably involve the Nrf2 pathway. The dual immunomodulatory and antimalarial mechanisms of curcumin action may mean that curcumin has potential as an adjuvant treatment limiting the risk of recrudescence following standard antimalarial therapy.

International audience; Bioreducible indolone-N-oxides have demonstrated their potent antimalarial activities with, however, a short half-life. The deox-ygenated analogues, 2-aryl-3H-indol-3-ones, a reduced chemical form obtained by synthesis, have similar antiplasmodial prop-erties against the blood stages of Plasmodium falciparumin vitro, with, however, a low aqueous solubility in vivo. Albumin-based nanosuspensions were used to solubilize these new compounds to conduct experiments in vivo using the P. berghei ANKA mu-rine model to determine the antimalarial activity of the most active derivative of this deoxygenated series, the 5-methoxy-2-(4-methoxyphenyl)-3H-indol-3-one (IND-1). Results were compared with its oxygenated analogue tested in the same conditions. Albumin-based nano-suspensions were prepared by precipitation, high-pressure homogenization and lyophilisation to yield nanoparticles of controlled diameter (350 nm) and polydispersity index (0.223) suitable for intravenous administration. The efficacy of IND-1, free and formulated in albumin-based nanosuspensions, was evaluated on a murine model infected with P. berghei ANKA. Assays revealed that the nanoformulation is much more potent (ED50 7.3 mg/kg/day, ip) than the non-formulated IND-1 (ED50 49.5 mg/kg/day, ip). Moreover, the albumin-based formulated nanosuspensions of IND-1 showed 100 % parasite-mia inhibition when administered iv and ip at 50 and 100 mg/kg/day, respectively. This inhibition was not reached with the clas-sical solvent/cosolvent methods. In conclusion, the albumin-based nanosuspensions of 2-aryl-3H-indol-3-ones greatly enhanced their antimalarial activities in vivo with a 7-fold decrease of the ED50 on ip administration compared to the free compound. How-ever, the reduction of the NO moiety did not significantly improve in vivo properties of the IND-1 compound compared, in the same conditions, to the INOD-1 compound.

Background Transmission of the malaria parasite Plasmodium falciparum from humans to the mosquito vector requires differentiation of a sub-population of asexual forms replicating within red blood cells into non-dividing male and female gametocytes. The nature of the molecular mechanism underlying this key differentiation event required for malaria transmission is not fully understood. Methods Whole genome sequencing was used to examine the genomic diversity of the gametocyte non-producing 3D7-derived lines F12 and A4. These lines were used in the recent detection of the PF3D7_1222600 locus (encoding PfAP2-G), which acts as a genetic master switch that triggers gametocyte development. Results The evolutionary changes from the 3D7 parental strain through its derivatives F12 (culture-passage derived cloned line) and A4 (transgenic cloned line) were identified. The genetic differences including the formation of chimeric var genes are presented. Conclusion A genomics resource is provided for the further study of gametocytogenesis or other phenotypes using these parasite lines. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1254-1) contains supplementary material, which is available to authorized users.