Figures & data
Figure 1. Electron micrographs of the fibrils formed by (A) MSP2 8–15 peptide, (B) MSP2 1–25 peptide and (C) recombinant full-length FC27 MSP2.
Peptides and protein were incubated in PBS at 1 mg/ml. Samples were negatively stained and viewed using transmission electron microscopy. The scale bar represents 100 nm.
Reproduced with permission from [Citation6] © European Peptide Society and John Wiley & Sons, Ltd. (2007).
![Figure 1. Electron micrographs of the fibrils formed by (A) MSP2 8–15 peptide, (B) MSP2 1–25 peptide and (C) recombinant full-length FC27 MSP2.Peptides and protein were incubated in PBS at 1 mg/ml. Samples were negatively stained and viewed using transmission electron microscopy. The scale bar represents 100 nm.Reproduced with permission from [Citation6] © European Peptide Society and John Wiley & Sons, Ltd. (2007).](/cms/asset/703ff9c0-da7d-4f7a-aaf1-34e74fe9121a/ifso_a_12363807_f0001.jpg)
Figure 2. Longitudinal section of a Plasmodium knowlesi merozoite showing the bristly appearance of the cell coat which covers the entire surface.
In this example, the parasite is attached apically to a red cell by a few long thread-like projections.
Image reprinted with kind permission from [Citation10]. Springer Science+Business Media B.V.
![Figure 2. Longitudinal section of a Plasmodium knowlesi merozoite showing the bristly appearance of the cell coat which covers the entire surface.In this example, the parasite is attached apically to a red cell by a few long thread-like projections.Image reprinted with kind permission from [Citation10]. Springer Science+Business Media B.V.](/cms/asset/06d021fe-5cc6-4892-b0a6-e3c2a21ce877/ifso_a_12363807_f0002.jpg)
Figure 3. Prediction of amyloid-forming sequences in the MSP2 isoforms 3D7 and FC27. Highlighted in yellow are the sequences with strong consensus prediction according to FoldAmyloid, Tango, Waltz, Aggrescan and Zhang's method algorithms.
The arrowheads indicate trypsin cleaving sites.
Figure 4. Atomic force microscope images of aggregates of MSP2-derived peptides (digested or not with trypsin) formed after incubation in PBS at 37°C for 2–3 weeks.
The images were obtained using a Dimension 3100 atomic force microscope (Veeco Instruments, Inc., CA, USA) and NP-S probes (Veeco) with nominal spring constants of 0.06 N/m to scan the samples in tapping mode in liquid at 0.5–1.0 Hz scan rates. Highly oriented pyrolytic graphite (NT-MDT Co., Moscow, Russia) was used as a substrate.
Figure 6. Adhesion of parasitized red blood cells and of free Plasmodium falciparum parasites to an in vitro blood–brain barrier model.
HBMECs were initially expanded in a 75-cm2 flask precoated with fibronectin. Plasmodium falciparum strain E8B was grown in vitro in group B human erythrocytes using previously described conditions [Citation26]. For the selection of cytoadherent parasites, pRBCs were passed over human umbilical vein endothelial cells (HUVECs) in two selection rounds. Late trophozoite- and schizont-containing pRBCs were selected in 70% Percoll and seeded at approximately 2 × 107 pRBCs/cm2 on an HUVEC monolayer. The co-culture model of the BBB was adapted from a previously described protocol [Citation27]. Briefly, rat astrocytes (RA) obtained from 2-day newborn rats were initially seeded at 60,000 cells/well onto a poly-D-lysine-coated plate. After 4 days, HBMECs were seeded at 16,500 cells/well onto collagen-coated 0.4 µm pore membrane inserts. This EC monolayer separated the system into an apical (bloodstream) and basolateral (brain tissue) compartments. The growth medium was changed every 3 days until cells reached confluence and tight junctions appeared. On the 9th HBMECs-RA co-culture day, pRBCs were purified in 70% Percoll, stained with wheat germ agglutinin-tetramethylrhodamine conjugate, and seeded at 1 × 106 cells/well onto membrane inserts (apical compartment of the BBB model containing the EC layer). The system was co-cultured overnight at 37°C in a humidified atmosphere with 5% CO2 (protocol adapted from [Citation28]). The day after, HBMECs and pRBCs were fixed and stained for the analysis by confocal microscopy of the tight junction marker protein zonula occludens-1 (ZO-1) [Citation29]. Cell nuclei were stained with DAPI, and pRBCs were specifically detected with a monoclonal antibody against schizont-infected RBCs [Citation26]. Arrowheads in the Merge panel indicate individual P. falciparum merozoites.
![Figure 6. Adhesion of parasitized red blood cells and of free Plasmodium falciparum parasites to an in vitro blood–brain barrier model.HBMECs were initially expanded in a 75-cm2 flask precoated with fibronectin. Plasmodium falciparum strain E8B was grown in vitro in group B human erythrocytes using previously described conditions [Citation26]. For the selection of cytoadherent parasites, pRBCs were passed over human umbilical vein endothelial cells (HUVECs) in two selection rounds. Late trophozoite- and schizont-containing pRBCs were selected in 70% Percoll and seeded at approximately 2 × 107 pRBCs/cm2 on an HUVEC monolayer. The co-culture model of the BBB was adapted from a previously described protocol [Citation27]. Briefly, rat astrocytes (RA) obtained from 2-day newborn rats were initially seeded at 60,000 cells/well onto a poly-D-lysine-coated plate. After 4 days, HBMECs were seeded at 16,500 cells/well onto collagen-coated 0.4 µm pore membrane inserts. This EC monolayer separated the system into an apical (bloodstream) and basolateral (brain tissue) compartments. The growth medium was changed every 3 days until cells reached confluence and tight junctions appeared. On the 9th HBMECs-RA co-culture day, pRBCs were purified in 70% Percoll, stained with wheat germ agglutinin-tetramethylrhodamine conjugate, and seeded at 1 × 106 cells/well onto membrane inserts (apical compartment of the BBB model containing the EC layer). The system was co-cultured overnight at 37°C in a humidified atmosphere with 5% CO2 (protocol adapted from [Citation28]). The day after, HBMECs and pRBCs were fixed and stained for the analysis by confocal microscopy of the tight junction marker protein zonula occludens-1 (ZO-1) [Citation29]. Cell nuclei were stained with DAPI, and pRBCs were specifically detected with a monoclonal antibody against schizont-infected RBCs [Citation26]. Arrowheads in the Merge panel indicate individual P. falciparum merozoites.](/cms/asset/121f88fd-7fda-4a23-a411-ae215aff1707/ifso_a_12363807_f0006.jpg)
Table 1. Average scores obtained using the Waltz algorithm for the prediction of amyloid regions in MSP2 GPI anchor and signal peptides and in the two amyloidogenic stretches of the amyloid β peptide.