Plasmodium cynomolgi is the closest relative of P. vivax and the primary experimental model for relapsing malaria, hypnozoite biology, and blood-stage drug susceptibility. Yet existing reference genomes remain fragmented, leaving structurally complex, AT-rich regions largely unresolved. We generated a chromosome-scale genome assembly for the K4-A7 cloned line of P. cynomolgi Berok by combining Hi-C chromosome conformation capture, Oxford Nanopore long reads, PacBio, and Illumina sequencing. The assembly spans 14 chromosomes plus mitochondrial and apicoplast genomes, with only seven unplaced minor contigs, the fewest for any non- P. falciparum Plasmodium genome, and an N50 of 3.06 Mb. Critically, this hybrid strategy resolved approximately 8 Mb of extremely AT-rich (~20% GC) sequence onto chromosomes 4, 8, and 13, anchoring what were previously unplaced or absent contigs into a continuous chromosomal framework. These subtelomere-like expansions (SLEs) constitute ~26.5% of the chromosomal genome and are enriched for PIR/VIR, STP1, variable surface antigen, and methyltransferase pseudogene families. Despite low gene density, SLE-encoded genes are transcriptionally active and show stage-specific expression across the erythrocytic cycle. Integrated lifecycle transcriptomics across 7,006 genes revealed a ~54-hour erythrocytic cycle with a "just-in-time" transcriptional cascade closely resembling that of P. vivax . Phylogenomic analyses and pairwise amino acid comparisons across more than 2,600 single-copy orthologs show that Berok forms a deeply diverged P. cynomolgi lineage, suggesting a distinct subspecies. This assembly establishes a high-resolution genomic foundation for comparative malaria biology, drug discovery, and the study of subtelomeric architecture, host adaptation, and lineage boundaries in primate Plasmodium .

Fusarihexins C-E, a group of cyclodepsipeptides that contain a characteristic 2-hydroxy-4-methyl-pentanoic acid (HICA) residue, were recently isolated from the endophytic fungus Fusarium sp. The absolute stereochemistry of HICA was determined to be R in fusarihexin C using a modified Mosher's method. It was assumed that HICA in fusarihexins D and E would have the same configuration, as they are derived from the same biological source. Herein, we report the first total synthesis of the proposed structures of fusarihexins D and E as well as three new analogues. The compounds were synthesized by employing solid phase peptide synthesis (SPPS) and high dilution macrolactamization and characterized by NMR spectroscopy and high-resolution mass spectrometry (HRMS). Comparing the 1H and 13C NMR spectra of synthesized and natural compounds revealed that the HICA residue has the S-configuration in fusarihexin D. The antiplasmodium activity on Plasmodium falciparum and antitumor activity against MCF7 and A431 cells were also investigated. Encouragingly, fusarihexin D (with S-HICA) displayed potent antiplasmodium activity by interfering with the ring stage of the Plasmodium falciparum parasite life cycle (IC50 at 650 nM).

Artemisinin has long been a first-line antimalarial. Yet, its mode of action is still poorly understood. Emergence of artemisinin-resistant strains highlight the importance of addressing this question so as to develop better drugs and overcome resistance. In this study, we performed RNA-sequencing and proteomics studies on artemisinin treated parasites indicated a striking difference in the codon-usage pattern of differentially translated genes. Using a liquid chromatography-coupled mass spectrometry (LC-MS)-based platform, we have quantified the full spectrum of modified ribonucleosides on tRNA in P. falciparum in response to the drug. We found that N6-threonyl-carbomyladenosine (t6A), a universal tRNA modification found at position 37 is hypomodified in response to artemisinin induced stress. Additionally, we also found that artemisinin treatment resulted in a downregulation of PfSua5, an enzyme involved in the t6A biosynthesis machinery. These findings provide new insights into how artemisinin works. More broadly, the findings exposes the tRNA epitranscriptome as a vulnerability in the parasite that can be exploited for new drugs.

Sudden environmental changes are a recurring challenge for unicellular organisms, but a necessity for many to progress through their lifecycle. To transmit from its human host to mosquito vector, malaria parasites differentiate into male and female, semi-quiescent stages that can re-initiate development within seconds after transmission. Here, we identify the RNA modification N6-methyladenosine (m6A) as the mediator of a rapid, sex-specific, and temperature-sensitive mechanism to restructure protein synthesis during transmission. We find that male parasites maintain high levels of translation during their semi-quiescence that are rapidly repressed following mosquito uptake. This translational shutdown is essential for the continuation of male parasite development and depends on the m6A-binding protein YTH.2. We further show that m6A and YTH.2 are already present prior to transmission, but that their repressive interaction requires a temperature drop accompanying the exit from the human host. Hence, m6A appears to prime the parasite transcriptome and subsequently converts an environmental shift into a rapid translational response. ### Competing Interest Statement The authors have declared no competing interest.

Plasmodium falciparum artemisinin (ART) resistance is driven by mutations in kelch-like protein 13 (PfK13). Quiescence, a key aspect of resistance, may also be regulated by a yet unidentified epigenetic pathway. Transfer RNA modification reprogramming and codon bias translation is a conserved epitranscriptomic translational control mechanism that allows cells to rapidly respond to stress. We report a role for this mechanism in ART-resistant parasites by combining tRNA modification, proteomic and codon usage analyses in ring-stage ART-sensitive and ART-resistant parasites in response to drug. Post-drug, ART-resistant parasites differentially hypomodify mcm5s2U on tRNA and possess a subset of proteins, including PfK13, that are regulated by Lys codon-biased translation. Conditional knockdown of the terminal s2U thiouridylase, PfMnmA, in an ART-sensitive parasite background led to increased ART survival, suggesting that hypomodification can alter the parasite ART response. This study describes an epitranscriptomic pathway via tRNA s2U reprogramming that ART-resistant parasites may employ to survive ART-induced stress.

Ribonucleoside modifications comprising the epitranscriptome are present in all organisms and all forms of RNA, including mRNA, rRNA and tRNA, the three major RNA components of the translational machinery. Of these, tRNA is the most heavily modified and the tRNA epitranscriptome has the greatest diversity of modifications. In addition to their roles in tRNA biogenesis, quality control, structure, cleavage, and codon recognition, tRNA modifications have been shown to regulate gene expression post-transcriptionally in prokaryotes and eukaryotes, including humans. However, studies investigating the impact of tRNA modifications on gene expression in the malaria parasite Plasmodium falciparum are currently scarce. Current evidence shows that the parasite has a limited capacity for transcriptional control, which points to a heavier reliance on strategies for posttranscriptional regulation such as tRNA epitranscriptome reprogramming. This review addresses the known functions of tRNA modifications in the biology of P. falciparum while highlighting the potential therapeutic opportunities and the value of using P. falciparum as a model organism for addressing several open questions related to the tRNA epitranscriptome.

It is widely recognized that Plasmodium merozoites secrete ligands that interact with RBC receptors. Meanwhile the question on whether these interactions trigger RBC signals essential for invasion remains unresolved. There is evidence that Plasmodium falciparum parasites manipulate native RBC Ca2+ signaling to facilitate invasion. Here, we demonstrate a key role of RBC Ca2+ influx that is conserved across different Plasmodium species during invasion. RH5-basigin interaction triggers RBC cAMP increase to promote Ca2+ influx. The RBC signaling pathways can be blocked by a range of inhibitors during Plasmodium invasion, providing the evidence of a functionally conserved host cAMP-Ca2+ signaling that drives invasion and junction formation. Furthermore, RH5-basigin binding induces a pre-existing multimeric RBC membrane complex to undergo increased protein association containing the cAMP-inducing β-adrenergic receptor. Our work presents evidence of a conserved host cell signaling cascade necessary for Plasmodium invasion and will create opportunities to therapeutically target merozoite invasion.

In the absence of an efficacious vaccine, chemotherapy remains crucial to prevent and treat malaria. Given its key role in haemoglobin degradation, falcilysin constitutes an attractive target. Here, we reveal the mechanism of enzymatic inhibition of falcilysin by MK-4815, an investigational new drug with potent antimalarial activity. Using X-ray crystallography, we determine two binary complexes of falcilysin in a closed state, bound with peptide substrates from the haemoglobin α and β chains respectively. An antiparallel β-sheet is formed between the substrate and enzyme, accounting for sequence-independent recognition at positions P2 and P1. In contrast, numerous contacts favor tyrosine and phenylalanine at the P1’ position of the substrate. Cryo-EM studies reveal a majority of unbound falcilysin molecules adopting an open conformation. Addition of MK-4815 shifts about two-thirds of falcilysin molecules to a closed state. These structures give atomic level pictures of the proteolytic cycle, in which falcilysin interconverts between a closed state conducive to proteolysis, and an open conformation amenable to substrate diffusion and products release. MK-4815 and quinolines bind to an allosteric pocket next to a hinge region of falcilysin and hinders this dynamic transition. These data should inform the design of potent inhibitors of falcilysin to combat malaria. The antimalarial drug MK-4815 shifts falcilysin to a closed state inhibiting its proteolytic cycle, which provides insights for designing potent falcilysin inhibitors to combat malaria.

About 247 million cases of malaria occurred in 2021 with Plasmodium falciparum accounting for the majority of 619,000 deaths. In the absence of a widely available vaccine, chemotherapy remains crucial to prevent, treat, and contain the disease. The efficacy of several drugs currently used in the clinic is likely to suffer from the emergence of resistant parasites. A global effort to identify lead compounds led to several initiatives such as the Medicine for Malaria Ventures (MMV), a repository of compounds showing promising efficacy in killing the parasite in cell-based assays. Here, we used mass spectrometry coupled with cellular thermal shift assay to identify putative protein targets of MMV000848, a compound with an in vitro EC50 of 0.5 mu M against the parasite. Thermal shift assays showed a strong increase of P. falciparum purine nucleoside phosphorylase (PfPNP) melting temperature by up enzymatic assays returned a KD of 1.52 +/- 0.495 mu M and an IC50 value of 21.5 +/- 2.36 mu M. The inhibition is competitive with respect to the substrate, as confirmed by a cocrystal structure of PfPNP bound with MMV000848 at the active site, determined at 1.85 & Aring; resolution. In contrast to transition states inhibitors, MMV000848 specifically inhibits the parasite enzyme but not the human ortholog. An isobologram analysis shows subadditivity with immucillin H and with quinine respectively, suggesting overlapping modes of action between these compounds. These results point to PfPNP as a promising antimalarial target and suggest avenues to improve inhibitor potency.

Malaria drug resistance is hampering the fight against the deadliest parasitic disease affecting over 200 million people worldwide. We recently developed quinoline-quinazoline-based inhibitors (as compound 70) as promising new antimalarials. Here, we aimed to investigate their mode of action by using thermal proteome profiling (TPP). The eukaryotic translation initiation factor 3 (EIF3i) subunit I was identified as the main target protein stabilized by compound 70 in Plasmodium falciparum. This protein has never been characterized in malaria parasites. P. falciparum parasite lines were generated expressing either a HA tag or an inducible knockdown of the PfEIF3i gene to further characterize the target protein. PfEIF3i was stabilized in the presence of compound 70 in a cellular thermal shift Western blot assay, pointing that PfEIF3i indeed interacts with quinoline-quinazoline-based inhibitors. In addition, PfEIF3i-inducible knockdown blocks intra-erythrocytic development in the trophozoite stage, indicating that it has a vital function. We show that PfEIF3i is mostly expressed in late intra-erythrocytic stages and localizes in the cytoplasm. Previous mass spectrometry reports show that PfEIF3i is expressed in all parasite life cycle stages. Further studies will explore the potential of PfEIF3i as a target for the design of new antimalarial drugs active all along the life cycle of the parasite.

The scale and duration of neutralizing antibody responses targeting SARS-CoV-2 viral variants represents a critically important serological parameter that predicts protective immunity for COVID-19. In this study, we describe the development and employment of a new functional assay that measures neutralizing antibodies for SARS-CoV-2 and present longitudinal data illustrating the impact of age, sex and comorbidities on the kinetics and strength of vaccine-induced antibody responses for key variants in an Asian volunteer cohort. We also present an accurate quantitation of serological responses for SARS-CoV-2 that exploits a unique set of in-house, recombinant human monoclonal antibodies targeting the viral Spike and nucleocapsid proteins and demonstrate a reduction in neutralizing antibody titres across all groups 6 months post-vaccination. We also observe a marked reduction in the serological binding activity and neutralizing responses targeting recently newly emerged Omicron variants including XBB 1.5 and highlight a significant increase in cross-protective neutralizing antibody responses following a third dose (boost) of vaccine. These data illustrate how key virological factors such as immune escape mutations combined with host demographic factors such as age and sex of the vaccinated individual influence the strength and duration of cross-protective serological immunity for COVID-19.

Plasmodium parasites contribute to one of the highest global infectious disease burdens. To achieve this success, the parasite has evolved a range of specialized subcellular compartments to extensively remodel the host cell for its survival. The information to fully understand these compartments is likely hidden in the so far poorly characterized Plasmodium species spatial proteome. To address this question, we determined the steady-state subcellular location of more than 12,000 parasite proteins across five different species by extensive subcellular fractionation of erythrocytes infected by Plasmodium falciparum, Plasmodium knowlesi, Plasmodium yoelii, Plasmodium berghei, and Plasmodium chabaudi. This comparison of the pan-species spatial proteomes and their expression patterns indicates increasing species-specific proteins associated with the more external compartments, supporting host adaptations and post-transcriptional regulation. The spatial proteome offers comprehensive insight into the different human, simian, and rodent Plasmodium species, establishing a powerful resource for understanding species-specific host adaptation processes in the parasite.

The complex life cycle of the human malaria parasite, Plasmodium falciparum, is driven by specific transcriptional programs, but it is unclear how most genes are activated or silenced at specific times. There is an association between transcription and spatial organization; however, the molecular mechanisms behind genome organization are unclear. While P. falciparum lacks key genome-organizing proteins found in metazoans, it has all core components of the cohesin complex. To investigate the role of cohesin in P. falciparum, we functionally characterize the cohesin subunit Structural Maintenance of Chromosomes protein 3 (SMC3). SMC3 knockdown during early stages of the intraerythrocytic developmental cycle (IDC) upregulates a subset of genes involved in erythrocyte egress and invasion, which are normally expressed at later stages. ChIP-seq analyses reveal that during the IDC, SMC3 enrichment at the promoter regions of these genes inversely correlates with gene expression and chromatin accessibility. These data suggest that SMC3 binding contributes to the repression of specific genes until their appropriate time of expression, revealing a new mode of stage-specific gene repression in P. falciparum.

Complement Receptor Type 1 ( CR1 ) is a malaria-associated gene that encodes a transmembrane receptor of erythrocytes and is crucial for malaria parasite invasion. The expression of CR1 contributes to the rosetting of erythrocytes in the brain bloodstream, causing cerebral malaria, the most severe form of the disease. Here, we study the history of adaptation against malaria by analyzing selection signals in the CR1 gene. We used whole-genome sequencing datasets of 907 healthy individuals from malaria-endemic and non-endemic populations. We detected robust positive selection in populations from the hyperendemic regions of East India and Papua New Guinea. Importantly, we identified a new adaptive variant, rs12034598, which is associated with a slower rate of erythrocyte sedimentation and is linked with a variant associated with low levels of CR1 expression. The combination of the variants likely drives natural selection. In addition, we identified a variant rs3886100 under positive selection in West Africans, which is also related to a low level of CR1 expression in the brain. Our study shows the fine-resolution history of positive selection in the CR1 gene and suggests a population-specific history of CR1 adaptation to malaria. Notably, our novel approach using population genomic analyses allows the identification of protective variants that reduce the risk of malaria infection without the need for patient samples or malaria individual medical records. Our findings contribute to understanding of human adaptation against cerebral malaria.

The most virulent human malaria parasite, Plasmodium falciparum , has a complex life cycle between its human host and mosquito vector. Each stage is driven by a specific transcriptional program, but with a relatively high ratio of genes to specific transcription factors, it is unclear how genes are activated or silenced at specific times. The P. falciparum genome is relatively euchromatic compared to the mammalian genome, except for specific genes that are uniquely heterochromatinized via HP1. There seems to be an association between gene activity and spatial organization; however, the molecular mechanisms behind genome organization are unclear. While P. falciparum lacks key genome-organizing proteins found in metazoans, it does have all core components of the cohesin complex. In other eukaryotes, cohesin is involved in sister chromatid cohesion, transcription, and genome organization. To investigate the role of cohesin in P. falciparum , we combined genome editing, mass spectrometry, chromatin immunoprecipitation and sequencing (ChIP-seq), and RNA sequencing to functionally characterize the cohesin subunit Structural Maintenance of Chromosomes protein 3 (SMC3). SMC3 knockdown in early stages of the intraerythrocytic developmental cycle (IDC) resulted in significant upregulation of a subset of genes involved in erythrocyte egress and invasion, which are normally expressed at later stages. ChIP-seq of SMC3 revealed that over the IDC, enrichment at the promoter regions of these genes inversely correlates with their expression and chromatin accessibility levels. These data suggest that SMC3 binding helps to repress specific genes until their appropriate time of expression, revealing a new mode of stage-specific, HP1-independent gene repression in P. falciparum .

Surveillance of SARS-CoV-2 infection is critical for controlling the current pandemic. Antigen rapid tests (ARTs) provide a means for surveillance. Available lateral flow assay format ARTs rely heavily on nitrocellulose paper, raising challenges in supply shortage. Vertical flow assay (VFA) with cellulose paper as test material attracts much attention as a complementary test approach. However, current reported VFAs are facing challenges in reading the test signal from the bottom face of the test cassette, complicating the test workflow and hindering translation into rapid test application. Here, we address this gap with an enhanced VFA against SARS-CoV-2 N protein that adapts a cellulose pull-down test format allowing (1) one-step sample application at the top of the test cassette and (2) readout of the test signal from the top. We also demonstrate the feasibility of translating the enhanced VFA into a point-of-care application that can help in SARS-CoV-2 surveillance.

Successful Plasmodium falciparum merozoite invasion requires the activation of red blood cell (RBC) signalling pathways. The binding of parasite ligand reticulocyte binding protein homologue 5 (RH5) to its host receptor Basigin is essential for merozoite invasion and triggers a Ca 2+ influx in RBCs. Here we observed that RH5-bound RBCs form a multimeric protein complex containing Basigin, CD44 and β2-adrenergic receptor (β2AR), suggesting that RH5-Basigin interaction is functionally associated with the host cAMP signalling pathway. Interestingly, we detected a characteristic rise in cAMP levels in the RBC upon RH5-Basigin interaction, which can be blocked by G protein and cAMP-synthesising adenylyl cyclase (AC) inhibitors. Furthermore, we demonstrated that RBC L-type Ca 2+ channel inhibitor and cAMP signalling inhibitors are able to block merozoite invasion. Checkerboard invasion inhibition assay containing different combinations of signalling inhibitors also exhibited a drastic amplification of inhibition levels, indicating that these signalling proteins are functioning in a common signalling cascade to activate the L-type Ca 2+ channels. Taken together, this study provides new insights into the role of a host cAMP-Ca 2+ signalling pathway during merozoite invasion and sheds new light on antimalarial therapeutic strategies to tackle the high infection rate and growing threat of drug resistant parasites. Key Points A pre-existing Basigin-associated membrane protein complex undergoes increased protein assembly upon RH5 binding on the RBC surface. Plasmodium falciparum merozoite exploits host cAMP signalling to initiate Ca 2+ influx in the RBC.

Malaria parasite-Plasmodium falciparum infection is a severe healthcare problem in tropical and subtropical regions. Early detection of such disease is vital in reducing mortality and controlling the spread in the affected areas. In this article, detecting the ring-stage malaria parasite is carried out by using the proposed photoacoustic (PA)-surface acoustic wave (SAW) sensing and detection system. By optical radiation, the PA signal is generated from the red blood cell (RBC), and then, the SAW sensor could transfer the acoustic wave into the electrical signal for the analysis. The SAW sensor is manufactured via the standard two-step lithography techniques. The proposed system consists of a tunable pulse laser device with the adjusting optical components, a PA-SAW sensor, a function generator, two analog front-end (AFE) circuits, a digital oscilloscope, a workstation for data postprocessing, and several auxiliary coaxial cables for data communications. With the acquired PA signal and its fast Fourier transform (FFT) spectrum from the various specimens, the cultured ring-stage malaria parasite in the infected whole blood could be detected at a low concentration level of 0.5% with the PA-SAW sensor. The proposed system demonstrates its excellent potential in the early diagnosis of the malaria parasite.