Discovery of small molecule entry inhibitors targeting the linoleic acid binding pocket of SARS-CoV-2 spike protein.

Spike glycoprotein has a significant role in the entry of SARS-CoV-2 to host cells, which makes it a potential drug target. Continued accumulation of non-synonymous mutations in the receptor binding domain of spike protein poses great challenges in identifying antiviral drugs targeting this protein. This study aims to identify potential entry inhibitors of SARS-CoV-2 using virtual screening and molecular dynamics (MD) simulations from three distinct chemical libraries including Pandemic Response Box, Drugbank and DrugCentral, comprising 6971 small molecules. The molecules were screened against a binding pocket identified in the receptor-binding domain (RBD) region of the spike protein which is known as the linoleic acid binding pocket, a highly conserved motif among several SARS-CoV-2 variants. Through virtual screening and binding free energy calculations, we identified four top-scoring compounds, MMV1579787 ([2-Oxo-2-[2-(3-phenoxyphenyl)ethylamino]ethyl]phosphonic acid), Tretinoin, MMV1633963 ((2E,4E)-5-[3-(3,5-dichlorophenoxy)phenyl]penta-2,4-dienoic acid) and Polydatin, which were previously reported to have antibacterial, antifungal or antiviral properties. These molecules showed stable binding on MD simulations over 100 ns and maintained stable interactions with TYR365, PHE338, PHE342, PHE377, TYR369, PHE374 and LEU368 of the spike protein RBD that are found to be conserved among SARS-CoV-2 variants. Our findings were further validated with free energy landscape, principal component analysis and dynamic cross-correlation analysis. Our in silico analysis of binding mode and MD simulation analyses suggest that the identified compounds may impede viral entrance by interacting with the linoleic acid binding site of the spike protein of SARS-CoV-2 regardless of its variants, and they thus demand for further in vitro and in vivo research.Communicated by Ramaswamy H. Sarma.

Search for novel Plasmodium falciparum PfATP4 inhibitors from the MMV Pandemic Response Box through a virtual screening approach.

Owing to its life cycle involving multiple hosts and species-specific biological complexities, a vaccine against Plasmodium, the causative agent of Malaria remains elusive. This makes chemotherapy the only viable means to address the clinical manifestations and spread of this deadly disease. However, rapid surge in antimalarial resistance poses significant challenges to our efforts to eliminate Malaria since the best drug available to-date; Artemisinin and its combinations are also rapidly losing efficacy. Sodium ATPase (PfATP4) of Plasmodium has been recently explored as a suitable target for new antimalarials such as Cipargamin. Prior studies showed that multiple compounds from the Medicines for Malaria Venture (MMV) chemical libraries were efficient PfATP4 inhibitors. In this context, we undertook a structure- based virtual screening approach combined to Molecular Dynamic (MD) simulations to evaluate whether new molecules with binding affinity towards PfATP4 could be identified from the Pandemic Response Box (PRB), a 400-compound library of small molecules launched in 2019 by MMV. Our analysis identified new molecules from the PRB library that showed affinity for distinct binding sites including the previously known G358 site, several of which are clinically used anti-bacterial (MMV1634383, MMV1634402), antiviral (MMV010036, MMV394033) or antifungal (MMV1634494) agents. Therefore, this study highlights the possibility of exploiting PRB molecules against Malaria through abrogation of PfATP4 activity.Communicated by Ramaswamy H. Sarma.

Optical diffraction tomography and image reconstruction to measure host cell alterations caused by divergent Plasmodium species.

Malaria is a life-threatening infectious disease caused by parasites of the genus Plasmodium. Understanding the biological features of various parasite forms is important for the optical diagnosis and defining pathological states, which are often constrained by the lack of ambient visualization approaches. Here, we employ a label-free tomographic technique to visualize the host red blood cell (RBC) remodeling process and quantify changes in biochemical properties arising from parasitization. Through this, we provide a quantitative body of information pertaining to the influence of host cell environment on growth, survival, and replication of P. falciparum and P. vivax in their respective host cells: mature erythrocytes and young reticulocytes. These exquisite three-dimensional measurements of infected red cells demonstrats the potential of evolving 3D imaging to advance our understanding of Plasmodium biology and host-parasite interactions.

High-Content Phenotypic Screen of a Focused TCAMS Drug Library Identifies Novel Disruptors of the Malaria Parasite Calcium Dynamics.

The search for new antimalarial drugs with unexplored mechanisms of action is currently one of the main objectives to combat the resistance already in the clinic. New drugs should target specific mechanisms that once initiated lead inevitably to the parasite's death and clearance and cause minimal toxicity to the host. One such new mode of action recently characterized is to target the parasite's calcium dynamics. Disruption of the calcium homeostasis is associated with compromised digestive vacuole membrane integrity and release of its contents, leading to programmed cell death-like features characterized by loss of mitochondrial membrane potential and DNA degradation. Intriguingly, chloroquine (CQ)-treated parasites were previously reported to exhibit such cellular features. Using a high-throughput phenotypic screen, we identified 158 physiological disruptors (hits) of parasite calcium distribution from a small subset of approximately 3000 compounds selected from the GSK TCAMS (Tres Cantos Anti-Malarial Set) compound library. These compounds were then extensively profiled for biological activity against various CQ- and artemisinin-resistant Plasmodium falciparum strains and stages. The hits were also examined for cytotoxicity, speed of antimalarial activity, and their possible inhibitory effects on heme crystallization. Overall, we identified three compounds, TCMDC-136230, -125431, and -125457, which were potent in inducing calcium redistribution but minimally inhibited heme crystallization. Molecular superimposition of the molecules by computational methods identified a common pharmacophore, with the best fit assigned to TCMDC-125457. There were low cytotoxicity or CQ cross-resistance issues for these three compounds. IC50 values of these three compounds were in the low micromolar range. In addition, TCMDC-125457 demonstrated high efficacy when pulsed in a single-dose combination with artesunate against tightly synchronized artemisinin-resistant ring-stage parasites. These results should add new drug options to the current armament of antimalarial drugs as well as provide promising starting points for development of drugs with non-classical modes of action.

Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells.

More than one-third of the world's population is exposed to Plasmodium vivax malaria, mainly in Asia1. P. vivax preferentially invades reticulocytes (immature red blood cells)2-4. Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. 5) and the reticulocyte-binding proteins (PvRBPs)6-10. PvRBP2b binds to the transferrin receptor CD71 (ref. 11), which is selectively expressed on immature reticulocytes12. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71+) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.

A deep learning approach to the screening of malaria infection: Automated and rapid cell counting, object detection and instance segmentation using Mask R-CNN.

Accurate and early diagnosis is critical to proper malaria treatment and hence death prevention. Several computer vision technologies have emerged in recent years as alternatives to traditional microscopy and rapid diagnostic tests. In this work, we used a deep learning model called Mask R-CNN that is trained on uninfected and Plasmodium falciparum-infected red blood cells. Our predictive model produced reports at a rate 15 times faster than manual counting without compromising on accuracy. Another unique feature of our model is its ability to generate segmentation masks on top of bounding box classifications for immediate visualization, making it superior to existing models. Furthermore, with greater standardization, it holds much potential to reduce errors arising from manual counting and save a significant amount of human resources, time, and cost.

Epstein-Barr virus infection modulates blood-brain barrier cells and its co-infection with Plasmodium falciparum induces RBC adhesion.

Plasmodium falciparum infection-mediated Epstein-Barr virus (EBV) reactivation is well established in malaria-endemic countries. We hypothesize that, during malaria onset, the reactivated EBV can infect human brain microvascular endothelial cells (HBECs). This may cause severe cerebral manifestations. We infected HBECs with EBV in vitro. The subsequent gene expression pattern of EBV, inflammatory and endothelial markers was analysed using qRT-PCR. Further, a wound-healing assay for cells maintaining blood-brain barrier (BBB) integrity was performed to investigate the effect of EBV-infected HBECs secretions. The RBC adhesion assay was conducted to assess RBC attachment onto HBECs during EBV and P. falciparum mono- and co-infection. Our experiments revealed that EBV infection of HBECs significantly elevated several inflammatory (TNFα, CCL2) and endothelial (integrin ß3, PECAM, VEGFA, VWF, claudin-5, cx37) markers. The EBV-infected HBECs secretion significantly reduced migration of HBECs, glial and neuronal cells. Additionally, EBV-P. falciparum co-infection significantly (P < 0.05) enhanced RBC adhesion to HBECs compared to mono-infection scenarios. Conclusively, the EBV infection of HBECs led to endothelial activation and modulated the BBB microenvironment. The EBV-P. falciparum co-infection scenario increased RBC adhesion on ECs which is a hallmark of cerebral malaria. Together with malaria, EBV infection can aid in exacerbation of cerebral malaria pathology.

Comparing infrared spectroscopic methods for the characterization of Plasmodium falciparum-infected human erythrocytes.

Malaria, caused by parasites of the species Plasmodium, is among the major life-threatening diseases to afflict humanity. The infectious cycle of Plasmodium is very complex involving distinct life stages and transitions characterized by cellular and molecular alterations. Therefore, novel single-cell technologies are warranted to extract details pertinent to Plasmodium-host cell interactions and underpinning biological transformations. Herein, we tested two emerging spectroscopic approaches: (a) Optical Photothermal Infrared spectroscopy and (b) Atomic Force Microscopy combined with infrared spectroscopy in contrast to (c) Fourier Transform InfraRed microspectroscopy, to investigate Plasmodium-infected erythrocytes. Chemical spatial distributions of selected bands and spectra captured using the three modalities for major macromolecules together with advantages and limitations of each method is presented here. These results indicate that O-PTIR and AFM-IR techniques can be explored for extracting sub-micron resolution molecular signatures within heterogeneous and dynamic samples such as Plasmodium-infected human RBCs.

Exosome Purification and Analysis Using a Facile Microfluidic Hydrodynamic Trapping Device.

Exosomes are nanosized (30-150 nm) extracellular vesicles (EVs) secreted by various cell types. They are easily accessible in biological fluids and contain specific disease biomarkers, making them attractive for diagnosis and prognosis applications. Accurate biological characterization of exosomes is an important step toward clinical applications that require effective and precise isolation of subpopulations of exosomes. It is therefore of particular importance to develop an efficient and reliable exosome purification technique to isolate exosomes from the heterogeneous extracellular fluids. In this work, we intend to isolate and visualize exosomes by combining an affinity-based method and passive microfluidic particle trapping. Microbeads with a diameter of 20 µm are first functionalized with streptavidin and biotinylated antibodies and then used to immobilize and enrich exosomes on their surfaces using antigen-antibody affinity binding. We have developed a microfluidic device with trapping arrays to efficiently trap a large number of individual microbeads with enriched exosomes at the single-particle level, i.e., one single bead per trapping site, on the basis of a passive hydrodynamic trapping principle. The large-scale microfluidic single-bead trapping permits massively multiplexed fluorescence detection and quantification of the individual beads, which prevents the optical interfering of background noise as well as allowing one to acquire an average fluorescence density of a single bead for an accurate fluorescence-based exosome quantification. In addition, on-chip elusion and lysis of the protein and RNA content of captured exosomes enable further molecular analysis of exosomes, including Western blot and quantitative polymerase chain reaction. This microfluidic device provides a rapid and straightforward capturing and quantification method to analyze EVs for a variety of biological studies and applications.

Whole-Cell Phenotypic Screening of Medicines for Malaria Venture Pathogen Box Identifies Specific Inhibitors of Plasmodium falciparum Late-Stage Development and Egress.

We report a systematic, cellular phenotype-based antimalarial screening of the Medicines for Malaria Venture Pathogen Box collection, which facilitated the identification of specific blockers of late-stage intraerythrocytic development of Plasmodium falciparum First, from standard growth inhibition assays, we identified 173 molecules with antimalarial activity (50% effective concentration [EC50] ≤ 10 µM), which included 62 additional molecules over previously known antimalarial candidates from the Pathogen Box. We identified 90 molecules with EC50 of ≤1 µM, which had significant effect on the ring-trophozoite transition, while 9 molecules inhibited the trophozoite-schizont transition and 21 molecules inhibited the schizont-ring transition (with ≥50% parasites failing to proceed to the next stage) at 1 µM. We therefore rescreened all 173 molecules and validated hits in microscopy to prioritize 12 hits as selective blockers of the schizont-ring transition. Seven of these molecules inhibited the calcium ionophore-induced egress of Toxoplasma gondii, a related apicomplexan parasite, suggesting that the inhibitors may be acting via a conserved mechanism which could be further exploited for target identification studies. We demonstrate that two molecules, MMV020670 and MMV026356, identified as schizont inhibitors in our screens, induce the fragmentation of DNA in merozoites, thereby impairing their ability to egress and invade. Further mechanistic studies would facilitate the therapeutic exploitation of these molecules as broadly active inhibitors targeting late-stage development and egress of apicomplexan parasites relevant to human health.

Evaluation of ferrocenyl phosphines as potent antimalarials targeting the digestive vacuole function of Plasmodium falciparum.

Owing to their lipophilic nature and chemical stability, ferrocene and its derivatives have been widely explored as antimicrobial agents, in combination with other active chemical 'war heads'. A prime example is ferroquine, an analogue of chloroquine obtained by covalently bonding ferrocene to 4-aminoquinoline, which possesses superior efficacy against multi-drug resistant malaria parasites. Herein, we explored the possibility of combining the ferrocenyl moiety with a phosphine unit and the subsequent inclusion of gold(i) to derive a molecular framework with demonstrated potential in inhibiting parasitic diseases. A library of 24 compounds consisting of 5 non-functionalized ferrocenyl enones and 19 ferrocenyl phosphine derivatives were synthesized, verified and tested against Plasmodium (P.) falciparum, which allowed us to identify compounds with low micromolar potency against both normal and chloroquine-resistant strains. Through flow cytometry combined with microscopic examination of Giemsa-stained thin smears, we observed that most of the active compounds interfered with trophozoite development as well as schizont maturation. The gold complex, namely G3, derived from the hydrophosphination of the terminal furan bearing an enone substrate showed the highest inhibitory potential. We demonstrate that G3 is affecting the parasite's metabolic processes as evident from the swollen digestive vacuole. Furthermore, G3 significantly affected heme de-toxification as determined through the ß-hematin assay, which caused apparent oxidative stress on parasites leading to death. Collectively, these results point out the potential of gold-conjugated ferrocenyl phosphine derivatives as antimalarials targeting the digestive vacuole function and metabolism of parasites.

A reference document on Permissible Limits for solvents and buffers during in vitro antimalarial screening.

Antimalarial drug discovery expands on targeted and phenotype-based screening of potential inhibitory molecules to ascertain overall efficacy, phenotypic characteristics and toxicity, prior to exploring pharmacological optimizations. Candidate inhibitors may have varying chemical properties, thereby requiring specific reconstitution conditions to ensure solubility, stability or bioavailability. Hence, a variety of solvents, buffers, detergents and stabilizers become part of antimalarial efficacy assays, all of which, above certain threshold could interfere with parasite viability, invasion or red blood cell properties leading to misinterpretation of the results. Despite their routine use across malaria research laboratories, there is no documentation on non-toxic range for common constituents including DMSO, glycerol, ethanol and methanol. We herein constructed a compatibility reference guide for 14 such chemicals and estimated their Permissible Limit against P. falciparum asexual stages at which viability and replication of parasites are not compromised. We also demonstrate that at the estimated Permissible Limit, red blood cells remain healthy and viable for infection by merozoites. Taken together, this dataset provides a valuable reference tool for the acceptable concentration range for common chemicals during in vitro antimalarial tests.

Febrile Temperature Elevates the Expression of Phosphatidylserine on Plasmodium falciparum (FCR3CSA) Infected Red Blood Cell Surface Leading to Increased Cytoadhesion.

During the asexual intra-erythrocytic cycle, Plasmodium (P.) falciparum exports parasitic proteins to the surface of infected red blood cells (iRBCs) facilitating its cytoadhesion to various endothelial host receptors. This adhesive behavior is a critical contributor towards disease manifestation. However, little is known about the influence of recurring elevated temperature - a common symptom of the malaria infection - on the adhesive properties of iRBCs to endothelial receptors. To address this, we performed dual-micropipette step-pressure technique between P. falciparum (strain FCR3CSA) iRBCs and Chinese Hamster Ovary cells expressing Chondroitin sulfate A (CHO-CSA) after transient iRBCs incubation at febrile temperatures which revealed increase in adhesion parameters. Furthermore, flow cytometry analysis revealed an increase in phosphatidylserine (PS) expression on the iRBC surface following exposure to febrile temperature. The adhesion between iRBCs and CHO-CSA cells was remarkably reduced in presence of soluble Annexin V, indicating the mediation of PS on the adhesion events. Our results suggest that elevated PS recruitment on iRBC under thermally stressed conditions contributes to the increased adhesive behavior of iRBCs CSA-binding phenotype to CHO-CSA.

Cytoskeleton Remodeling Induces Membrane Stiffness and Stability Changes of Maturing Reticulocytes.

Reticulocytes, the precursors of erythrocytes, undergo drastic alterations in cell size, shape, and deformability during maturation. Experimental evidence suggests that young reticulocytes are stiffer and less stable than their mature counterparts; however, the underlying mechanism is yet to be fully understood. Here, we develop a coarse-grained molecular-dynamics reticulocyte membrane model to elucidate how the membrane structure of reticulocytes contributes to their particular biomechanical properties and pathogenesis in blood diseases. First, we show that the extended cytoskeleton in the reticulocyte membrane is responsible for its increased shear modulus. Subsequently, we quantify the effect of weakened cytoskeleton on the stiffness and stability of reticulocytes, via which we demonstrate that the extended cytoskeleton along with reduced cytoskeleton connectivity leads to the seeming paradox that reticulocytes are stiffer and less stable than the mature erythrocytes. Our simulation results also suggest that membrane budding and the consequent vesiculation of reticulocytes can occur independently of the endocytosis-exocytosis pathway, and thus, it may serve as an additional means of removing unwanted membrane proteins from reticulocytes. Finally, we find that membrane budding is exacerbated when the cohesion between the lipid bilayer and the cytoskeleton is compromised, which is in accord with the clinical observations that erythrocytes start shedding membrane surface at the reticulocyte stage in hereditary spherocytosis. Taken together, our results quantify the stiffness and stability change of reticulocytes during their maturation and provide, to our knowledge, new insights into the pathogenesis of hereditary spherocytosis and malaria.

Targeted Phenotypic Screening in Plasmodium falciparum and Toxoplasma gondii Reveals Novel Modes of Action of Medicines for Malaria Venture Malaria Box Molecules.

The Malaria Box collection includes 400 chemically diverse small molecules with documented potency against malaria parasite growth, but the underlying modes of action are largely unknown. Using complementary phenotypic screens against Plasmodium falciparum and Toxoplasma gondii, we report phenotype-specific hits based on inhibition of overall parasite growth, apicoplast segregation, and egress or host invasion, providing hitherto unavailable insights into the possible mechanisms affected. First, the Malaria Box library was screened against tachyzoite stage T. gondii and the half-maximal effective concentrations (EC50s) of molecules showing ≥80% growth inhibition at 10 µM were determined. Comparison of the EC50s for T. gondii and P. falciparum identified a subset of 24 molecules with nanomolar potency against both parasites. Thirty molecules that failed to induce acute growth inhibition in T. gondii tachyzoites in a 2-day assay caused delayed parasite death upon extended exposure, with at least three molecules interfering with apicoplast segregation during daughter cell formation. Using flow cytometry and microscopy-based examinations, we prioritized 26 molecules with the potential to inhibit host cell egress/invasion during asexual developmental stages of P. falciparum. None of the inhibitors affected digestive vacuole integrity, ruling out a mechanism mediated by broadly specific protease inhibitor activity. Interestingly, five of the plasmodial egress inhibitors inhibited ionophore-induced egress of T. gondii tachyzoites. These findings highlight the advantage of comparative and targeted phenotypic screens in related species as a means to identify lead molecules with a conserved mode of action. Further work on target identification and mechanism analysis will facilitate the development of antiparasitic compounds with cross-species efficacy. IMPORTANCE The phylum Apicomplexa includes many human and animal pathogens, such as Plasmodium falciparum (human malaria) and Toxoplasma gondii (human and animal toxoplasmosis). Widespread resistance to current antimalarials and the lack of a commercial vaccine necessitate novel pharmacological interventions with distinct modes of action against malaria. For toxoplasmosis, new drugs to effectively eliminate tissue-dwelling latent cysts of the parasite are needed. The Malaria Box antimalarial collection, managed and distributed by the Medicines for Malaria Venture, includes molecules of novel chemical classes with proven antimalarial efficacy. Using targeted phenotypic assays of P. falciparum and T. gondii, we have identified a subset of the Malaria Box molecules as potent inhibitors of plastid segregation and parasite invasion and egress, thereby providing early insights into their probable mode of action. Five molecules that inhibit the egress of both parasites have been identified for further mechanistic studies. Thus, the approach we have used to identify novel molecules with defined modes of action in multiple parasites can expedite the development of pan-active antiparasitic agents.

High-Content Screening of the Medicines for Malaria Venture Pathogen Box for Plasmodium falciparum Digestive Vacuole-Disrupting Molecules Reveals Valuable Starting Points for Drug Discovery.

Plasmodium falciparum infections leading to malaria have severe clinical manifestations and high mortality rates. Chloroquine (CQ), a former mainstay of malaria chemotherapy, has been rendered ineffective due to the emergence of widespread resistance. Recent studies, however, have unveiled a novel mode of action in which low-micromolar levels of CQ permeabilized the parasite's digestive vacuole (DV) membrane, leading to calcium efflux, mitochondrial depolarization, and DNA degradation. These phenotypes implicate the DV as an alternative target of CQ and suggest that DV disruption is an attractive target for exploitation by DV-disruptive antimalarials. In the current study, high-content screening of the Medicines for Malaria Venture (MMV) Pathogen Box (2015) was performed to select compounds which disrupt the DV membrane, as measured by the leakage of intravacuolar Ca2+ using the calcium probe Fluo-4 AM. The hits were further characterized by hemozoin biocrystallization inhibition assays and dose-response half-maximal (50%) inhibitory concentration (IC50) assays across resistant and sensitive strains. Three hits, MMV676380, MMV085071, and MMV687812, were shown to demonstrate a lack of CQ cross-resistance in parasite strains and field isolates. Through systematic analyses, MMV085071 emerged as the top hit due to its rapid parasiticidal effect, low-nanomolar IC50, and good efficacy in triggering DV disruption, mitochondrial degradation, and DNA fragmentation in P. falciparum These programmed cell death (PCD)-like phenotypes following permeabilization of the DV suggests that these compounds kill the parasite by a PCD-like mechanism. From the drug development perspective, MMV085071, which was identified to be a potent DV disruptor, offers a promising starting point for subsequent hit-to-lead generation and optimization through structure-activity relationships.

Quantitative mass spectrometry of human reticulocytes reveal proteome-wide modifications during maturation.

Erythropoiesis is marked by progressive changes in morphological, biochemical and mechanical properties of erythroid precursors to generate red blood cells (RBC). The earliest enucleated forms derived in this process, known as reticulocytes, are multi-lobular and spherical. As reticulocytes mature, they undergo a series of dynamic cytoskeletal re-arrangements and the expulsion of residual organelles, resulting in highly deformable biconcave RBCs (normocytes). To understand the significant, yet neglected proteome-wide changes associated with reticulocyte maturation, we undertook a quantitative proteomics approach. Immature reticulocytes (marked by the presence of surface transferrin receptor, CD71) and mature RBCs (devoid of CD71) were isolated from human cord blood using a magnetic separation procedure. After sub-fractionation into triton-extracted membrane proteins and luminal samples (isobaric tags for relative and absolute quantitation), quantitative mass spectrometry was conducted to identify more than 1800 proteins with good confidence and coverage. While most structural proteins (such as Spectrins, Ankyrin and Band 3) as well as surface glycoproteins were conserved, proteins associated with microtubule structures, such as Talin-1/2 and ß-Tubulin, were detected only in immature reticulocytes. Atomic force microscopy (AFM)-based imaging revealed an extended network of spectrin filaments in reticulocytes (with an average length of 48 nm), which shortened during reticulocyte maturation (average spectrin length of 41 nm in normocytes). The extended nature of cytoskeletal network may partly account for increased deformability and shape changes, as reticulocytes transform to normocytes.

A portable image-based cytometer for rapid malaria detection and quantification.

Increasing resistance by malaria parasites to currently used antimalarials across the developing world warrants timely detection and classification so that appropriate drug combinations can be administered before clinical complications arise. However, this is often challenged by low levels of infection (referred to as parasitemia) and presence of predominantly young parasitic forms in the patients' peripheral blood. Herein, we developed a simple, inexpensive and portable image-based cytometer that detects and numerically counts Plasmodium falciparum infected red blood cells (iRBCs) from Giemsa-stained smears derived from infected blood. Our cytometer is able to classify all parasitic subpopulations by quantifying the area occupied by the parasites within iRBCs, with high specificity, sensitivity and negligible false positives (~ 0.0025%). Moreover, we demonstrate the application of our image-based cytometer in testing anti-malarial efficacy against a commercial flow cytometer and demonstrate comparable results between the two methods. Collectively, these results highlight the possibility to use our image-based cytometer as a cheap, rapid and accurate alternative for antimalarial testing without compromising on efficiency and minimal processing time. With appropriate filters applied into the algorithm, to rule out leukocytes and reticulocytes, our cytometer may also be used for field diagnosis of malaria.

P. falciparum RH5-Basigin interaction induces changes in the cytoskeleton of the host RBC.

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte-binding protein homologues (RHs) and erythrocyte-binding like proteins are two families involved in the invasion leading to merozoite-red blood cell (RBC) junction formation. Ca2+ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca2+ signaling, which triggers the erythrocyte-binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5-basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5-Basigin interaction on its own triggers a Ca2+ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca2+ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.