Antimalarial drug resistance poses a critical threat to global malaria control efforts. Despite the continuous development of novel antimalarial compounds, the emergence of drug resistance remains inevitable, highlighting the urgent need for paradigm-shifting therapeutic approaches. Here, we propose an innovative chimeric antigen receptor-macrophage (CAR-M) cell therapy that circumvents traditional small-molecule limitations by harnessing the innate phagocytic capacity of macrophages. This strategy exploits the specific adhesive interactions between Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and host receptors (CD36, ICAM-1/CD54, EPCR/CD201) to enable targeted recognition and elimination of infected erythrocytes. By engineering macrophages with chimeric antigen receptors directed against PfEMP1-binding domains, we establish a cell-based immunotherapy platform that provides sustained anti-parasitic activity independent of conventional drug susceptibility profiles. This approach represents a fundamental departure from chemical-based interventions by providing a potentially resistance-proof therapeutic modality for drug-resistant malaria.

The pathogenesis of Plasmodium falciparum malaria involves coordinated molecular events, including host cell invasion, intraerythrocytic replication cycles, and antigenic variation, which are critically dependent on the stage-specific regulation of parasite proteins. Despite their biological significance, the molecular governance of these pathogenic mechanisms remains inadequately characterized. Our investigation reveals that the expression dynamics of an ApiAP2 family transcription factor, PfAP2-V (PF3D7_1239200), exhibit strong temporal coordination with the transcriptional activation of virulence-associated genes during the blood-stage development of P. falciparum parasites. These genes encompass those coding for merozoite surface antigens and the inner membrane complex families and the var gene clusters encoding P. falciparum erythrocyte membrane protein 1 (PfEMP1) cytoadherence proteins. Conditional knockdown of pfap2-v expression significantly decreased parasite intraerythrocytic proliferation, resulted in structural abnormalities in infected erythrocyte surface topology, and markedly reduced the cytoadhesion capacity of infected erythrocytes to human endothelial receptors, such as CD36, ICAM-1, and PECAM-1/CD31. Mechanistic analyses revealed that PfAP2-V directly associates with specific chromosomal regions, where it is involved in chromatin interactions and facilitates transcriptional activation. These findings establish PfAP2-V as a master transcriptional regulator that governs both parasite proliferation and the expression of the virulence factors critical for P. falciparum malaria pathogenesis.

Artemisinin antimalarial drugs initially exhibited remarkable efficacy against Plasmodium falciparum. However, their poor solubility and low bioavailability necessitate high doses and lead to an extremely short in vivo half-life. These limitations not only drive the emergence of drug-resistant Plasmodium strains but also compromise long-term therapeutic outcomes. Herein, we report a zein-based sustained release formulation, wherein zein, a natural maize protein, serves as a biocompatible nanocarrier to effectively encapsulate artemisinin (ART). Notably, this nanocarrier formulation achieves a 200-fold enhancement in ART's water solubility, addressing a key bottleneck of ART-based therapies. In vitro assays confirm that the zein-based formulation allows for the sustained release of ART, which could help maintain therapeutic concentrations over extended periods and displayed different release rates and good dispersibility in both acid and basic environments. Importantly, in vitro evaluations also demonstrate that the nanoformulation exerts potent inhibitory effects against ART-resistant P. falciparum strains in both ring survival assay and recrudescence assay, attributed to the sustained maintenance of effective ART concentrations. In vivo studies, utilizing both rodent malaria models and humanized erythrocyte mouse models, further validate the nanoformulation's therapeutic potential. The zein nanocarrier significantly prolongs ART's in vivo half-life via its sustained-release capability, thereby maintaining effective blood concentrations over an extended duration. Compared to free ART, the nanoformulation exhibits superior efficacy in reducing parasitemia, preventing malaria recrudescence, and, most notably, overcoming ART resistance in drug-resistant Plasmodium infections. Collectively, these findings establish the zein-based nanocarrier as a promising strategy to optimize ART-based therapies by addressing solubility and pharmacokinetic limitations while effectively combating drug-resistant malaria. IMPORTANCE:Half of the world's population is at risk of malaria infection, and artemisinin (ART) turns out to be a powerful medicine for malaria control. The rapid emergence and global spread of resistance to ART have led to a significantly increasing clinical treatment failure rate worldwide. A critical limitation of ART is its extremely short blood half-life (~1 h), which results in rapid declines in plasma drug concentrations below therapeutic thresholds. Some parasites may switch into a "dormant" form, which is less sensitive to ART, resulting in recrudescence following treatment. Thus, developing a sustained-release formulation provides a promising solution to prolong the in vivo half-life of ART. Additionally, its relatively low solubility restricts its in vivo bioavailability, primarily due to the limited dissolution and absorption of the compound in aqueous biological environments. In this study, we prepared a zein-based sustained-release formulation of ART for oral and intraperitoneal administration. Our results indicate that this zein-based sustained release nanoformulation not only significantly improves ART's water solubility (a key barrier to its bioavailability) but also extends its in vivo half-life via controlled drug release. Importantly, the prolonged half-life ensures sustained therapeutic ART concentrations, directly enhancing the formulation's ability against ART-resistant P. falciparum strains. Collectively, these results highlight the formulation's substantial potential for clinical application in improving ART-based antimalarial therapy.

Abstract Schistosomiasis morbidity and mortality are primarily driven by egg-triggered granulomas with subsequent fibrosis. However, the dynamics of cellular composition within these granulomas, particularly the identity and function of key immune cells that orchestrate their formation and evolution, remain poorly understood. Here, we combine single-cell RNA sequencing with multiplex immunofluorescence to characterize the hepatic immune landscape in Schistosoma japonicum -infected mice and further validate the functional roles of two neutrophil subsets. We find that eggs alter hepatic immune cell composition, which is characterized by extensive neutrophil recruitment and differentiation. Neutrophil recruitment correlates with CXCL2 derived from both an autocrine loop and paracrine signaling from monocytes. Cellular localization analysis reveals that granulomas progress through three distinct developmental stages: early Ly6G hi F4/80 lo Desmin lo , developing Ly6G mid F4/80 hi Desmin mid , and advanced Ly6G lo F4/80 mid Desmin hi . Neutrophil subsets display a zonal distribution within granulomas, with CD177 + neutrophils surrounding the eggs and Ltf + neutrophils localizing to the mid-outer layer. Cd177 knockdown reduces granuloma size and fibrosis, whereas Ltf suppression increases these pathological features, indicating that CD177 and LTF exert opposite effects on granuloma formation. Our findings provide new insights into the cellular complexity of S. japonicum egg-induced granulomas and may help guide the development of novel therapies for liver fibrosis.

Cerebral malaria involves the expression of a particular var subgroup, termed upsA, in Plasmodium falciparum, which encodes a subset of PfEMP1 variants that avidly bind to human receptors on endothelial cells, resulting in obstruction of microvasculature in the brain and severe pathogenesis. Despite the clinical importance of the upsA var genes, the mechanism of their low-frequency activation in asymptomatic or uncomplicated malaria cases remains poorly understood. Here, we identify a JmjC-domain–containing histone demethylase PfJmjC1 as a vital transcriptional silencer of upsA var genes, which may negatively regulate malaria pathogenesis. Genome-wide profiling reveals that PfJmjC1 localizes to heterochromatic loci including the entire var gene family, and interacts with canonical heterochromatin factors such as HP1 at the schizont stage. However, PfJmjC1 preferentially binds to the promoter regions of upsA var loci in ring-stage parasites in which non-upsA vars are activated. Genetic deletion or conditional knockdown of PfJmjC1 selectively derepresses upsA var and disrupts the var expression “counting” mechanism, accompanied by increased H3K4me3 and H3K9ac, as well as decreased H3K9me3 modification at upsA var loci. The altered PfEMP1 expression patterns and acquisition of multiple endothelial receptor–binding phenotypes are experimentally verified at the single-cell level. Additionally, Hi-C-seq and three-dimensional genome modeling show that PfJmjC1 deficiency reorganizes nuclear heterochromatin architecture, repositioning upsA var loci toward a euchromatic environment. Together, these findings not only uncover a mechanism controlling the transcriptional activity of severe malaria-associated virulence genes but also provide a potential therapeutic target against severe malaria. Severe malaria is linked to upsA var gene expression, whose regulation remains unclear. Here, the authors identify JmjC1 as an epigenetic regulator of upsA var genes at the transcriptional level through chromatin and nuclear organization.

BACKGROUND:Artemisinin partial resistance (ART-R) has been confirmed in four sub-Saharan African countries since 2020, but evidence from Ethiopia is limited to molecular surveys without phenotypic confirmation. We aimed to determine whether ART-R met WHO criteria in Ethiopian Plasmodium falciparum populations during 2024-25, integrating day-3 parasite positivity after artemether-lumefantrine treatment, Pfkelch13 genotyping, and the ring-stage survival assay (RSA)0-3 h on culture-adapted field isolates. METHODS:We conducted a prospective, multisite, surveillance study at five sentinel health facilities in Ethiopia (Rama, Werkamba, Mehoni, Bako, and Metehara). Patients aged 6 months or older with uncomplicated P falciparum malaria confirmed by microscopy received artemether-lumefantrine according to bodyweight (six doses during 3 days). Pfkelch13 was genotyped by Nanopore sequencing and Pfhrp2/Pfhrp3 deletions were assessed by quantitative PCR. The RSA0-3 h was performed on culture-adapted field isolates. Follow-up occurred on days 0 and 3. The main outcome was day-3 parasite positivity rate (defined as microscopically detectable asexual P falciparum parasitaemia on day 3 after initiation of artemether-lumefantrine). This study is registered with ClinicalTrials.gov, NCT07527182 (completed). FINDINGS:Patients were enrolled from June 6 to Dec 8, 2024, during the 2024 P falciparum transmission season at all five sentinel sites and from July 1 to Nov 8, 2025, during the 2025 transmission season at Werkamba. 3207 febrile patients were assessed for malaria, of whom 2771 were excluded and 436 (14%) were P falciparum-positive on microscopy. 277 (64%) patients were enrolled, of whom 153 (55%) returned for the day-3 parasitological assessment and 124 (45%) were lost to follow-up. 172 (62%) of 277 patients were male and 105 (38%) were female. The median age was 20·0 years (IQR 10·0-30·0). 243 (88%) had P falciparum monoinfection by PCR and 34 (12%) had P falciparum and Plasmodium vivax co-infections undetected by microscopy at enrolment. The day-3 parasite positivity rate was 19·6% ([95% CI 14·1-26·6] in 30 of 153 patients with available data). 26 (9%) of 277 enrolled patients carried a validated Pfkelch13 mutation and were positive on day 3, exceeding the WHO 5% threshold for confirmation of ART-R. A strong age-dependent gradient was observed in a post-hoc analysis, with six (38%) of 16 patients younger than 5 years, 14 (35%) of 40 aged 5-15 years, and ten (10%) of 97 older than 15 years (p=0·00035). R622I was detected in 143 (53%) samples of 271 genotyped isolates. Carrying an R622I mutation was associated with day-3 positivity (26 [33%] of 79) in a univariate analysis (crude odds ratio [OR] 7·85 [95% CI 2·58-23·91]; p=0·0003). After adjustment, the association remained strong and independent (adjusted OR 9·96 [95% CI 3·39-36·35]; p<0·0001). Of 70 P falciparum field isolates on day 0 collected for culture adaptation, only six were successfully maintained. Five R622I isolates carried the Pfkelch13 R622I mutation and exceeded the 1% in vitro threshold for ART-R (mean survival rates of 1·41% [SD 0·27] for EW04, 2·69% [0·90] for EW14, 1·07% [0·52] for EW23, 3·76% [0·35] for EW38, and 1·29% [0·04] for EW56). Pooled with the wild-type strains, all five R622I isolates exceeded the 1% threshold compared with three wild-type isolates that did not exceed this threshold (p=0·018). Pfhrp3 was the most frequent deletion (39·8% [95% CI 32·6-47·4]; in 66 of 166 patients), followed by double Pfhrp2/Pfhrp3 deletion (23·5% [17·7-30·5]; in 39), wild-type (23·5% [17·7-30·5]; in 39), and Pfhrp2 deletion (13·3% [8·9-19·3]; in 22). R622I prevalence was similar across all four deletion categories (range 42-56%; p=0·47). INTERPRETATION:Ethiopia is the fifth sub-Saharan African country now meeting WHO confirmation criteria for ART-R. High R622I prevalence and double deletion rates, consistent with distinct selective pressures, represent a dual threat to treatment and HRP2-based diagnosis of P falciparum malaria in this region. FUNDING:Fondation pour la Recherche Médicale, Institut Universitaire de France, Agence Nationale de la Recherche, Université de Strasbourg, and the National Natural Science Foundation of China. TRANSLATION:For the Amharic translation of the abstract see Supplementary Material section.

Schistosomiasis morbidity and mortality are primarily driven by hepatic granulomas triggered by parasite eggs with subsequent periportal fibrosis. As a common pathological consequence of various chronic liver injuries, hepatic fibrosis is well-documented. However, the dynamics of cellular composition within the schistosome egg-induced granuloma, particularly the identity and function of key immune cell subsets that orchestrate its formation and evolution, remain poorly resolved. To address this, we combined single-cell RNA sequencing with multiplex immunofluorescence, histopathological analysis, and serological profiling to comprehensively characterize the hepatic immune landscape in a murine model of Schistosoma japonicum infection, and further validated the functional roles of two neutrophil subsets through targeted marker gene knockdown. We found that egg deposition altered hepatic immune cell composition, characterized by extensive neutrophil recruitment and differentiation. Neutrophil recruitment correlated with CXCL2 derived from both an autocrine loop and paracrine signaling by monocytes. Cellular localization analysis of the developmental trajectory of granulomas showed that they progressed from the early Ly6G hi F4/80 lo Desmin lo stage, through the developing Ly6G mid F4/80 hi Desmin mid stage, to the advanced Ly6G lo F4/80 mid Desmin hi stage. In addition, neutrophil subsets display zonal distribution within early granulomas: CD177 + neutrophils surrounded the eggs, while Ltf + neutrophils localized to the mid-outer layer. Cd177 knockdown reduced granuloma size and fibrosis, while Ltf suppression increased these pathological features, indicating that CD177 + and LTF + neutrophils have opposite effects on granuloma formation. Our findings provide new insight into the cellular complexity of S. japonicum egg-induced granulomas and could help guide the development of novel treatments for liver fibrosis.

Abstract Background Plasmodium falciparum merozoite invasion of erythrocytes is an essential step in the asexual blood-stage cycle and a major target for antimalarial intervention. Rhoptry neck proteins play key roles in the formation and function of the tight junction, yet many remain poorly characterized. RALP1, a conserved rhoptry neck-associated leucine zipper-like protein, has been proposed to participate in erythrocyte binding and invasion. Conventional gene disruption attempts have been unsuccessful, suggesting that RALP1 may be essential for parasite survival. Nevertheless, its precise role and broader molecular impact during intraerythrocytic development remain to be fully elucidated. Methods We generated a 3 × HA-tagged conditional knockdown line (ralp1-ha-glmS) using CRISPR-Cas9-mediated homologous recombination. RALP1 abundance and subcellular localization were evaluated by Western blotting and immunofluorescence assays. Effects on parasite growth, schizont maturation, merozoite invasion, and merozoite numbers were assessed using tightly synchronized cultures and established invasion and cytological assays. Transcriptomic changes following GlcN-induced RALP1 knockdown were analyzed by RNA-seq at early ring and schizont stages. Sequence-based structural and epitope features were examined using IUPred2A, ANCHOR2, AlphaFold3, NetMHCpan, and NetMHCIIpan. Results Precise integration of the ha-glmS cassette enabled GlcN-inducible reduction of RALP1 protein levels, most prominently in schizonts. RALP1 knockdown reduced parasite proliferation, impaired schizont maturation, decreased merozoite numbers, and lowered erythrocyte invasion efficiency. RNA-seq showed limited effects in early rings but widespread downregulation of invasion- and host-parasite interaction-related genes in schizonts after correction for glucosamine-responsive transcripts, with GO enrichment highlighting processes related to host cell interaction, biological adhesion, and membrane-associated components. Sequence-based analyses indicated that RALP1 contains extensive intrinsically disordered regions with multiple predicted interaction motifs, while predicted B- and T-cell epitope hotspots concentrated within the C-terminal RBC-binding domain. AlphaFold3 modeling yielded low global confidence (pTM = 0.23), consistent with a primarily disordered architecture. Conclusions RALP1 is required for normal schizont maturation and efficient erythrocyte invasion in P. falciparum. Its partial knockdown perturbs transcription of key invasion ligands and apical components, indicating a broader role in preparing merozoites for host-cell entry. The extensive disorder, epitope-rich C-terminal region, and essential function of RALP1 highlight its potential as a candidate for therapeutic or vaccine targeting. Graphical Abstract

Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.

BACKGROUND:Neoadjuvant chemoimmunotherapy (NACI) shows promise for locally advanced cervical cancer (LACC), but drug-tolerant persister (DTP) cells and immunosuppressive microenvironmental adaptations limit clinical efficacy. The underlying determinants governing heterogeneous responses to NACI regimens remain poorly understood, particularly regarding how dynamic tumor-immune interactions shape therapeutic outcomes. METHODS:We characterized microenvironmental dynamics in patients with LACC by integrating single-cell RNA sequencing (RNA-seq), single-cell VDJ sequencing (n=10, five paired pre-NACI/post-NACI samples) and spatial transcriptomics (ChiCTR2300072535). Pathological response was assessed using major pathological response criteria. The findings were validated in an independent NACI cohort (n=23 with RNA-seq), multiplex immunohistochemistry (mIHC) analysis of six surgically resected specimens, as well as functional in vitro and murine models. RESULTS:MPR patients exhibited cytotoxic revival via oligoclonal expansion of tumor-reactive CD8+T cell clones and CCR5-mediated myeloid-T cell crosstalk. Conversely, non-MPR tumors exhibited endoplasmic reticulum (ER) stress-adapted DTP cells with elevated ER stress signaling, accompanied by a deficiency in tumor-specific T-cell clone expansion and an accumulation of transforming growth factor beta receptor 2 (TGFBR2) + myeloid DTP niches. Mechanistically, ER stress signaling via the inositol‑requiring enzyme 1 alpha (IRE1α) / X‑box binding protein 1 (XBP1) axis induces growth differentiation factor 15 (GDF15) production in DTP cells, contributing to treatment‑resistant microdomains. Pharmacological IRE1α inhibition synergized with chemoimmunotherapy to eradicate DTP populations in murine models. CONCLUSIONS:This study provides critical insights that NACI resistance stems from adaptive ER stress signaling in DTP cells and spatially organized immunosuppressive networks. Targeting the IRE1α/XBP1-GDF15 axis represents an actionable strategy to reprogram microenvironmental ecology and improve immunotherapy outcomes.

Introduction:Plasmodium falciparum, the causative agent of severe malaria, predominantly reproduces through asexual stages within human red blood cells, with a small subset differentiating into transmissible gametocytes. TCF25 is recognized in other eukaryotes as a protein with dual roles: a transcriptional regulator and a key component of the Ribosome-associated Quality Control (RQC) complex. Nevertheless, the precise biological function of TCF25 in Plasmodium spp. remains inadequately elucidated. Methods:To investigate the function of TCF25, we created a tcf25 knockout (tcf25_ko) parasite strain and conducted comparative transcriptomic analysis during the ring and schizont stages. Gametocyte induction experiments were performed to investigate the impact of tcf25 deletion on gametocyte development. Chromatin immunoprecipitation sequencing (ChIP-seq) was utilized to delineate the genome-wide binding profiles in schizont-stage parasites. Additionally, RT-qPCR was used to quantify changes in rRNA expression levels after tcf25 knockout. Results:Transcriptomic analysis of tcf25_ko parasites indicated substantial dysregulation, with 168 genes downregulated and 24 genes upregulated during the ring stage, and 53 genes downregulated and 4 genes upregulated during the schizont stage. ChIP-seq analysis identified 44 high-confidence TCF25-binding target genes, which notably included the rDNA. Furthermore, TCF25 deficiency resulted in upregulated rRNA expression, particularly affecting 28S rRNA, a core component of the 60S ribosomal subunit. Discussion:This study identifies TCF25 as a key regulator of various biological processes in P. falciparum. It is shown that TCF25 plays a crucial role in gametocytogenesis by influencing the ap2-g pathway. Additionally, a novel function of TCF25 in ribosomal biogenesis is uncovered, wherein it directly controls A-type rRNAs expression and ribosomal subunit homeostasis. These discoveries offer fresh perspectives on the molecular mechanisms that oversee transmission and ribosome biogenesis in malaria parasites.

BackgroundMalaria is caused by Plasmodium spp. and is a prevalent parasitic disease worldwide. To evade detection by the immune system, by switching variant gene expression, the malaria parasite continually establishes new patterns displaying a single variant erythrocyte surface antigen. The distinct surface molecules encoded by clonally variant gene families include var, rif, stevor, Pfmc-2tm, and surfins. However, the mechanism behind the exclusive expression of a single member of the variant gene family is still not clear. This study aims to describe the molecular process of variant gene switching from the perspective of the epitranscriptome, specifically by characterizing the role of the Plasmodium falciparum RNA m5C methyltransferase NSUN3.MethodsA conditional gene knockdown approach was adopted by incorporating the glucosamine-inducible glmS ribozyme sequence into the 3 ' untranslated region (UTR) of the pfnsun3 gene. A transgenic parasite line PfNSUN3-Ty1-Ribo was generated using CRISPR-Cas9 methods. The knockdown effect in the transgenic parasite was measured by a growth curve assay and western blot analysis. The transcriptome changes influenced by PfNUSN3 knockdown were detected by RNA sequencing (RNA-seq), and the direct RNA transcripts regulated by PfNUSN3 were validated by RNA immunoprecipitation and high-throughput sequencing (RIP-seq).ResultsGrowth curve analysis revealed that conditional knockdown of PfNSUN3 interfered with parasite growth. The parasitemia of the PfNSUN3 knockdown line showed a significant decline at the third round of the life cycle compared with the control line. The knockdown of PfNSUN3 altered the global transcriptome. RNA-seq analysis showed that at the ring-stage depletion of PfNSUN3 silenced almost all var genes, as well as the guanine/cytosine (GC)-rich non-coding RNA (ncRNA) ruf6 family. RNA RIP-seq arrays revealed that PfNSUN3 directly interacted with several var genes.ConclusionsOur findings demonstrate a vital role of PfNSUN3 in the process of the mutually exclusive expression of variant genes, and contribute to a better understanding of the complex mechanism of epigenetic regulation of gene expression in P. falciparum.

Egg granulomas caused by Schistosoma japonicum (S. japonicum) are important causes of morbidity and mortality in schistosomiasis. The intestine plays a crucial role in the complete life cycle of S. japonicum; eggs are transported through the intestine and excreted with feces. During this process, the interaction between the eggs and the intestine can trigger a strong intestinal immune system response and cause inflammation. Eggs in the intestine preferentially accumulate in Peyer’s patches (PPs). However, the cellular composition of intestinal granulomas and the impacts of egg deposition on the immune function of PPs remain poorly understood. Using a mouse model of S. japonicum infection, we revealed that the deposition of eggs disrupted the structure of PPs, resulting in immunosuppression. We further characterized the cellular composition of intestinal granulomas, revealing a layered distribution of neutrophils, macrophages, T cells, and B cells, with marked neutrophil accumulation. Single-cell RNA sequencing revealed that egg deposition drives B-cell apoptosis, T-cell exhaustion, and activation of fibrotic pathways in myeloid cells, collectively impairing PP function. In conclusion, the layered cellular architecture of intestinal granulomas in PPs suggests a unique immune microenvironment of egg-driven immunosuppression and fibrotic remodeling, and the identification of fibrotic pathways in myeloid cells provides a potential therapeutic target to alleviate fibrosis in patients with S. japonicum infection.

Artemisinin and its semisynthetic derivatives (ART) are crucial medicines in artemisinin-based combination therapies worldwide. Despite ART's efficacy, small proportions of young intraerythrocytic ring stage parasites can survive the drug's short half-life, and dormant forms can cause recrudescence if not cleared by partner drugs. Certain mutations in the Kelch propeller region of P. falciparum protein (PfK13) are linked to the higher ring-stage survival (RS), which above 1% can be a feature of 'artemisinin partial resistance'. Emerging evidence indicates epigenetic modulators may contribute to RS. Here, we report systematic evaluations of all putative histone acetyltransferases (HATs) of P. falciparum in 30 culture-adapted field isolates and 43 subcloned field isolates. Only PfMYST shows a full association with RS phenotype modulations. Knockdown experiments confirm the linkage of Pfmyst expression to these modulations, with evidence of altered metabolic processes. Through single-cell RNA sequencing, ChIP-seq analysis, and CRISPR/cas9 genetic manipulation, PfMYST-targeted RS-related genes have been identified and functionally validated. Multi-omics analysis indicates significant interplay of PfMYST and PfK13 mechanisms in RS. PfMYST epigenetic modulation extends to other antimalarials, including amodiaquine, pyrimethamine, chloroquine, and pyronaridine. Collectively, our findings provide important information on the epigenetic regulatory mechanism of P. falciparum RS after pulses of ART and other antimalarials.

Obligate intracellular parasites must efficiently invade host cells to complete their life cycle and facilitate transmission. For the malaria-causing parasite Plasmodium falciparum, the invasion of an erythrocyte is a critical process, and thereby a key target for intervention strategies. In this study, we investigate the role of the ApiAP2 family transcription factor PfAP2-06B (PF3D7_0613800) in the intraerythrocytic developmental cycle of P. falciparum and focus on its regulation of genes involved in erythrocyte invasion. Conditional knockdown of PfAP2-06B resulted in a defect in asexual growth and impaired erythrocyte invasion. Bulk RNA sequencing (RNA-seq) analysis revealed that PfAP2-06B modulates the expression of invasion-related genes during the schizont stage. Single-cell RNA sequencing indicated that PfAP2-06B influences invasion gene expression and contributes to stochastic variations in expression of cell-to-cell genes. These results underscore the critical function of PfAP2-06B in the process of erythrocyte invasion and suggest its potential as a target for novel malaria control strategies. Importance: Understanding gene regulation in Plasmodium falciparum is essential for uncovering mechanisms of parasite development and pathogenicity. The research underscores the pivotal role of PfAP2-06B in regulating critical aspects of Plasmodium intraerythrocytic development and host cell invasion, demonstrating that PfAP2-06B plays a key role in orchestrating stage-specific gene expression. These findings provide new insights into the transcriptional networks of P. falciparum and highlight PfAP2-06B as a potential target for therapeutic intervention. This work advances our understanding of malaria pathogenesis and developing effective interventions.

Purpose: To investigate the role of nitroquine (CI -679) against artemisinin-resistant Plasmodium falciparum (P. falciparum) C580Y strain. Methods: Antimalarial activity of CI -679 against blood stages in Plasmodium yoelii (P. yoelii) - infected BALB/c mice model was first identified. Thereafter, in vitro assays were performed to investigate the inhibitory activity against blood stages of artemisinin-sensitive P. falciparum 3D7 strain. Finally, the potential effect of CI -679 was also investigated on artemisinin-resistant P. falciparum, which was constructed by introducing C580Y mutation in K13 of the 3D7 using the CRISPR-CAS9 technology. Results: CI -679 significantly suppressed the growth of rodent malaria parasite, P. yoelii BY265, in a dose -dependent manner, and also inhibited the development of the parasites in mice (p < 0.05). Furthermore, CI -679 efficiently inhibited the growth of artemisinin-sensitive P. falciparum 3D7 in vitro, with more sensitivity against late phase of blood stages (p < 0.05). Also, CI -679 suppressed the development of artemisinin-resistant P. falciparum C580Y strain, and the inhibitory effect was comparable to that of artemisinin-sensitive 3D7 strain. Conclusion: CI -679 exhibits potent antimalarial activity against blood stages of P. yoelii BY265 in vivo, and both artemisinin-sensitive P. falciparum 3D7 and artemisinin-resistant P. falciparum C580Y in vitro. Further pharmacokinetic properties, tolerability and safety of the compound need to be investigated to support this claim.

RNA modifications (epitranscriptome) – such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ) – modulate RNA processing, stability, interaction, and translation, thereby playing critical roles in the development, replication, virulence, metabolism, and life cycle adaptations of parasitic protozoa. Here, we summarize potential homologs of the major human RNA modification regulatory factors in parasites, outline current knowledge on how RNA modifications affect parasitic protozoa, highlight the regulation of RNA modifications and their crosstalk, and discuss current progress in exploring RNA modifications as potential drug targets. This review contributes to our understanding of epitranscriptomic regulation of parasitic protozoa biology and pathogenesis and provides new perspectives for the treatment of parasitic diseases.

The tumor microenvironment (TME) directly determines patients' outcomes and therapeutic efficiencies. An in-depth understanding of the TME is required to improve the prognosis of patients with cervical cancer (CC). This study conducted single-cell RNA and TCR sequencing of six-paired tumors and adjacent normal tissues to map the CC immune landscape. T and NK cells were highly enriched in the tumor area and transitioned from cytotoxic to exhaustion phenotypes. Our analyses suggest that cytotoxic large-clone T cells are critical effectors in the antitumor response. This study also revealed tumor-specific germinal center B cells associated with tertiary lymphoid structures. A high-germinal center B cell proportion in patients with CC is predictive of improved clinical outcomes and is associated with elevated hormonal immune responses. We depicted an immune-excluded stromal landscape and established a joint model of tumor and stromal cells to predict CC patients' prognosis. The study revealed tumor ecosystem subsets linked to antitumor response or prognosis in the TME and provides information for future combinational immunotherapy.

Abstract The emergence and development of artemisinin resistance threaten global malaria control and elimination goals, thereby prompting research on the mechanisms of malaria parasite resistance. The mutation of Plasmodium falciparum Kelch 13 ( PfK13) protein is associated with artemisinin resistance, but the unique or common mechanism by which it leads to this resistance is unclear. By analyzing the different effects of PfK13 mutation on the P. falciparum transcriptome and proteome at the different stages, we found that PfK13 mutation did not significantly change glycolysis, TCA, pentose phosphate pathway (PPP) and oxidative phosphorylation but reduced the expression of reproduction- and DNA synthesis-related genes. Moreover, the reduced number of the merozoite, decreased amount of hemozoin, and slowed growth of P. falciparum 3D7C580Y were consistent with the changes, suggesting that the PfK13 mutation reduces hemoglobin ingestion, leading to artemisinin resistance, likely by decreasing the parasites' need for haem and iron. This study helps elucidate the mechanism of artemisinin resistance caused by the PfK13 mutation.