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BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.

The role of nitric oxide (NO) in the pathogenesis of cerebral malaria and its cognitive sequelae remains controversial. Cerebral malaria is still the worst complication of Plasmodium falciparum infection, which is characterized by high rates of morbidity and mortality. Even after recovery from infection due to antimalarial therapy, the development of cognitive impairment in survivors reinforces the need to seek new therapies that demonstrate efficacy in preventing long-lasting sequelae. During disease pathogenesis, reactive oxygen and nitrogen species (RONS) are produced after the established intense inflammatory response. Increased expression of the enzyme inducible nitric oxide synthase (iNOS) seems to contribute to tissue injury and the onset of neurological damage. Elevated levels of NO developed by iNOS can induce the production of highly harmful nitrogen-reactive intermediates such as peroxynitrite. To address this, we performed biochemical and behavioral studies in C57BL6 mice, aminoguanidine (specific pharmacological inhibitor of the enzyme iNOS) treated and iNOS−/−, infected with Plasmodium berghei ANKA (PbA), with the aim of clarifying the impact of iNOS on the pathogenesis of cerebral malaria. Our findings underscore the effectiveness of both strategies in reducing cerebral malaria and providing protection against the cognitive impairment associated with the disease. Here, the absence or blockade of the iNOS enzyme was effective in reducing the signs of cerebral malaria detected after six days of infection. This was accompanied by a decrease in the production of pro-inflammatory cytokines and reactive oxygen and nitrogen species. In addition, nitrotyrosine (NT-3), a marker of nitrosative stress, was also reduced. Futher, cognitive dysfunction was analyzed fifteen days after infection in animals rescued from infection by chloroquine treatment (25 mg/kg bw). We observed that both interventions on the iNOS enzyme were able to improve memory and learning loss in mice. In summary, our data suggest that the iNOS enzyme has the potential to serve as a therapeutic target to prevent cognitive sequelae of cerebral malaria. • The role of nitric oxide in cerebral malaria is a hotly debated, keeping the scientific communityintrigued. • Various sources of nitric oxide manifest unique effects on experimental cerebral malaria, sparking further research. • The immune system's production of nitric oxide, attributed to iNOS activity, can potentially induce tissue damage. • Nitric oxide derived from iNOS failed to mitigate vascular impairment associated with experimental cerebral malaria. • Therapies focused on iNOS inhibition hold promise in preventing cognitive sequelae due toexperimental cerebral malaria. [ABSTRACT FROM AUTHOR]

Background: Malaria, a disease caused by parasites of the genus Plasmodium, continues to impact many regions globally. The rise in resistance to artemisinin-based anti-malarial drugs highlights the need for new treatments. Ideally, new anti-malarials will kill the asymptomatic liver stages as well as the symptomatic blood stages. While blood stage screening assays are routine and efficient, liver stage screening assays are more complex and costly. To decrease the cost of liver stage screening, a previously reported luciferase detection protocol requiring only common laboratory reagents was adapted for testing against luciferase-expressing Plasmodium berghei liver stage parasites. Methods: After optimizing cell lysis conditions, the concentration of reagents, and the density of host hepatocytes (HepG2), the protocol was validated with 28 legacy anti-malarials to show this simple protocol produces a stable signal useful for obtaining quality small molecule potency data similar to that obtained from a high content imaging endpoint. The protocol was then used to screen the Global Health Priority Box (GHPB) and confirm the potency of hits in dose–response assays. Selectivity was determined using a galactose-based, 72 h HepG2 assay to avoid missing mitochondrial-toxic compounds due to the Crabtree effect. Receiver-operator characteristic plots were used to retroactively characterize the screens' predictive value. Results: Optimal luciferase signal was achieved using a lower HepG2 seed density (5 × 103 cells/well of a 384-well microtitre plate) compared to many previously reported luciferase-based screens. While producing lower signal compared to a commercial alternative, this luciferase detection method was found much more stable, with a > 3 h half-life, and robust enough for producing dose–response plots with as few as 500 sporozoites/well. A screen of the GHPB resulted in 9 hits with selective activity against P. berghei liver schizonts, including MMV674132 which exhibited 30.2 nM potency. Retrospective analyses show excellent predictive value for both anti-malarial activity and cytotoxicity. Conclusions: This method is suitable for high-throughput screening at a cost nearly 20-fold less than using commercial luciferase detection kits, thereby enabling larger liver stage anti-malarial screens and hit optimization make-test cycles. Further optimization of the hits detected using this protocol is ongoing. [ABSTRACT FROM AUTHOR]

Malaria remains a critical global health issue, particularly in tropical and subtropical regions. The disease, caused by Plasmodium parasites, is transmitted by Anopheles mosquitoes and can lead to severe complications and death if untreated. The emergence of drug‐resistant strains highlights the urgent need for new antimalarial agents. Gnetum gnemon, a plant native to Southeast Asia, has shown promise due to its rich bioactive compounds. This study aims to evaluate the suppressive, curative, and prophylactic antimalarial potential of Gnetum gnemon leaf extract (GGE) against Plasmodium berghei in mice. GGE was prepared using a combination of hot water extraction and microwave‐assisted heating. Acute toxicity tests revealed no significant adverse effects at a dose of 3000 mg/kg. The doses of 100, 200, and 400 mg/kg were selected based on preliminary toxicity assessments to systematically investigate the dose‐dependent antimalarial efficacy of the extract. Suppressive tests showed that GGE at doses of 100, 200, and 400 mg/kg significantly reduced parasitemia levels, with the highest dose achieving a 63.97% inhibition. In these tests, GGE also increased the mean survival time (MST) of treated mice compared to untreated controls. However, GGE did not exhibit significant curative effects, as parasitemia levels in the treated groups were similar to the untreated control group. Prophylactic tests indicated that GGE pretreatment did not significantly reduce parasitemia levels or improve MST compared to controls, unlike chloroquine (CQ), which demonstrated potent prophylactic efficacy with a significant increase in MST. These findings suggest that while GGE has notable suppressive antimalarial activity, it does not exhibit strong curative or prophylactic effects at the tested doses. This study contributes to the understanding of plant‐based antimalarial agents and underscores the importance of continued exploration of natural products for malaria treatment. [ABSTRACT FROM AUTHOR]

Plasmodium, the causative agent of malaria, infects hepatocytes prior to establishing a symptomatic blood stage infection. During this liver stage development, parasites reside in a parasitophorous vacuole (PV), whose membrane acts as the critical interface between the parasite and the host cell. It is well-established that host cell autophagy-related processes significantly impact the development of Plasmodium liver stages. Expression of genes related to autophagy and lysosomal biogenesis is orchestrated by transcription factor EB (TFEB). In this study, we explored the activation of host cell TFEB in Plasmodium berghei-infected cells during the liver stage of the parasite. Our results unveiled a critical role of proteins belonging to the Gamma-aminobutyric acid receptor-associated protein subfamily (GABARAP) of ATG8 proteins (GABARAP/L1/L2 and LC3A/B/C) in recruiting the TFEB-blocking FLCN-FNIP (Folliculin-Folliculin-interacting protein) complex to the PVM. Remarkably, the sequestration of FLCN-FNIP resulted in a robust activation of TFEB, reliant on conjugation of ATG8 proteins to single membranes (CASM) and GABARAP proteins. Our findings provide novel mechanistic insights into host cell signaling occurring at the PVM, shedding light on the complex interplay between Plasmodium parasites and the host cell during the liver stage of infection. Plasmodium berghei activates transcription factor EB by sequestering the host cell FLCN/FNIP complex to its parasitophorous vacuole membrane depending on host cell GABARAP proteins in a CASM dependent mechanism. [ABSTRACT FROM AUTHOR]

Salacia debilis Walp., (Celastraceae) is used traditionally in West Africa for the treatment of malaria. However, no scientific reports validating these effects and its active constituents are on record. Therefore, this study is aimed at evaluating the antimalarial effects, of its ethanolic extract and isolated compounds against Plasmodium falciparum 3D7 and P. berghei ANKA strains. Using chromatographic, spectrometric and spectroscopic techniques three compounds were isolated and characterised. The extract of S. debilis was active against P. falciparum 3D7, in an in vitro assay with IC50 of 12.0 ± 0.32 µg/ml. The three isolated compounds, namely 1,10-dihydroxy-6H-benzo[c]chromen-6-one (1), 8- hydroxy-3,4-dimethoxydibenzo[b,d]furan-1-carboxylic acid (2) and benzyl-2-methoxybenzoate (3), also showed antimalarial activity against Plasmodium berghei ANKA strain in curative and suppressive in vivo assays. The ethanolic extract and isolated compounds of S. debilis possess antimalarial effects. The isolated compounds may be responsible, at least in part, for the observed activities of the extract. [ABSTRACT FROM AUTHOR]

High mobility group box (HMGB) proteins belong to the high mobility group (HMG) superfamily of non-histone nuclear proteins that are involved in chromatin remodeling, regulation of gene expression, and DNA repair. When extracellular, HMGBs serve as alarmins inducing inflammation, and this is attributed to the proinflammatory activity of box B. Here, we show that Plasmodium HMGB1 has key amino acid changes in box B resulting in the loss of TNF-α stimulatory activity. Sitedirected mutagenesis of the critical amino acids in box B with respect to mouse HMGB1 renders recombinant Plasmodium berghei (Pb) HMGB1 capable of inducing TNF-a release. Targeted deletion of PbHMGB1 and a detailed in vivo phenotyping show that PbHMGB1 knockout (KO) parasites can undergo asexual stage development. Interestingly, Balb/c mice-infected with PbHMGB1KO parasites display a protective phenotype with subsequent clearance of blood parasitemia and develop long-lasting protective immunity against the challenges performed with Pb wildtype parasites. The characterization of splenic responses shows prominent germinal centers leading to effective humoral responses and enhanced T follicular helper cells. There is also complete protection from experimental cerebral malaria in CBA/CaJ mice susceptible to cerebral pathogenesis with subsequent parasite clearance. Transcriptomic studies suggest the involvement of PbHMGB1 in pir expression. Our findings highlight the gene regulatory function of parasite HMGB1 and its in vivo significance in modulating the host immune responses. Further, clearance of asexual stages in PbHMGB1KO-infected mice underscores the important role of parasite HMGB1 in host immune evasion. These findings have implications in developing attenuated bloodstage vaccines for malaria. [ABSTRACT FROM AUTHOR]

Objectives: Investigating how nanoparticle systems interact in whole blood (WB) is critical to evaluating the effectiveness of malaria therapy. Methods: We decided to establish a pharmacokinetic/pharmacodynamic (PK/PD) model of the quinine population in WB using Plasmodium berghei-infected mice, with a subsequent model comparison for nanocapsules coated with polysorbate (NCP80) or prepared with Eudragit® RS (NCEUD). The WB quinine population pharmacokinetic model in rats was developed using plasma and partition coefficients for rat erythrocytes. Mouse WB quinine population PK/PD modeling was developed using allometrically scaled literature-free mouse quinine pharmacokinetic data and covariate values to obtain a WB population pharmacokinetic model for quinine and nanocapsules in mice. This allowed for PK/PD modeling of the quinine population with the WB concentration and parasitemia data in mice. All models were built in NONMEN. Results: The WB quinine concentration profiles in rats were characterized using a two-compartment model. Nanoencapsulation reduced clearance and central compartment volume and increased peripherical compartimental volume. A maximum effect model described the PK/PD of the quinine WB population in mice, demonstrating that NCEUD enhances the antimalarial effect. Conclusions: Quinine WB is a good surrogate for describing the response to exposure in malaria. NCEUD outperformed NCP80 and free quinine, suggesting that cationic surfaces improve the potential for treating malaria. [ABSTRACT FROM AUTHOR]

Malaria treatment is burdened by many challenges including growing resistance to currently used antimalarial drugs. Thus, the continuous search for safe and effective remedies is necessary. This study investigated the antiplasmodial activities of the methanol extract (ME) of Phoenix reclinata and its n-hexane (HF), ethyl acetate (EF), and butanol (BF) fractions. Phytochemistry of the extract and fractions was conducted. The effect of methanol extract (100, 200, and 400 mg/kg) and its solvent fractions (200 and 400 mg/kg) on parasitaemia levels was evaluated using curative, suppressive, and prophylactic anti-plasmodial models. The antipyretic effect of its solvent fractions was also determined. In the curative model, ME exhibited dose-dependent chemosuppression of 18.4%, 46.4%, and 79.6% at 100, 200, and 400 mg/kg. At 200 and 400 mg/kg, HF produced 24.5% and 28.9%, EF produced 56.8% and 85.3%, and BF produced 48% and 56.8%. In the Suppressive model, ME exhibited a dose-dependent chemosuppression of 19.08%, 45.23%, and 80% by 100, 200, and 400 mg/kg, HF exhibited 15.55% and 28.89%, EF exhibited 61.32% and 81.94%, BF exhibited 34.06% and 43.08% for 200 and 400 mg/kg. In the prophylactic model, ME exhibited a dose-dependent chemosuppression of 8.71 %, 15.59%, and 17.48 % by 100, 200, and 400 mg/kg. HF exhibited 2.66% and 8.6% for 200 and 400 mg/kg. BF exhibited anti-plasmodial suppression of 15.73% and 21.68% for 200 and 400 mg/kg. EF exhibited the highest chemosuppression in all the models. [ABSTRACT FROM AUTHOR]

Although malaria is curable, it remains the leading cause of mortality in Nigeria. Eliminating malaria is a pressing concern due to the challenge of drug-resistant Plasmodium parasites. Therefore, there is an urgent need for antimalarial agents that are affordable, safe, and effective against drug-resistant strains. The antimalarial potential of Salix ledermannii ethanol leaf extracts and their effect on PCV and haemoglobin levels, as well as the phytochemical compositions of the extract, were evaluated in this study using Plasmodium berghei infected mice as models. 0.2 ml Distilled water (Dw) was used as the negative control, while 5 mg/kg chloroquine and 1.2 mg/kg pyrimethamine served as the positive control. The phytochemical composition was analysed using standard chemical tests and GC-Ms techniques. Compared to the negative control, S. ledermannii showed significant chemo-suppression at 100 mg (44.38 %) and 300 mg/kg (46.75 %). Also, 5 mg/kg-chloroquine and treatment at 300 mg/kg significantly inhibited parasitaemia compared to the control group in the curative test model (p<0.05). The prophylactic test did not differ significantly (p>0.05) across treatment groups. The crude extract contained alkaloids, tannins, steroids, terpenes, anthraquinones, phenols, and saponins, and GC-Ms analysis revealed forty-three (43) known compounds, with benzoic acid (27.21%), phenol (15.22%), ß-DGlucopyranose (12.29%), Salicylalcohol (11.88%), 1-Butanol, 3-methyl (6.93%), Catechol (6.35%) and -ethyl-5,6-dihydo-2H-pyran-2-one (5.48%) having the highest concentrations. S. ledermannii poses no adverse effect on PCV and H.B. levels of treated mice and exhibits significant antimalarial properties. Thus, this plant can serve as a novel lead for compounds in the next-generation antimalarial drugs. [ABSTRACT FROM AUTHOR]

Liver-stage genetically attenuated malaria parasites (GAPs) are powerful immunogens that provide protection against sporozoite challenge. We previously generated two late liver-stage-arresting GAPs by deleting the stearoyl-CoA desaturase (Scd) or sporozoite conserved orthologous transcript 1 (Scot1) genes in Plasmodium berghei. Immunization with Scd or Scot1 GAP conferred complete protection against a sporozoite challenge. In a safety study, we observed rare breakthrough blood-stage infections in mice inoculated with high doses of sporozoites, indicating that both GAPs were incompletely attenuated. In this study, we generated a Scd/Scot1 GAP by dual gene deletion. This resulted in complete attenuation of the parasites in the liver and did not transition to blood-stage infection despite a high-dose sporozoite challenge. The Scd/Scot1 KO and WT GFP parasites were indistinguishable during blood, mosquito and early liver stage development. Moreover, Scd/Scot1 KO liver-stage schizonts exhibited an abnormal apicoplast biogenesis and nuclear division phenotype, failed to form hepatic merozoites, and exhibited late liver-stage arrest. Compared with early-arresting Speld KO immunization, late-stage liver-arresting Scd/Scot1 KO induces greater and broader CD8+ T-cell responses and elicits stage-transcending immunity that provides superior protection in C57BL/6 mice. These data prove that multiple gene deletions lead to complete attenuation of the parasite and support the development of late liver stage-arresting P. falciparum GAP. [ABSTRACT FROM AUTHOR]

Malaria parasites hijack the metabolism of their mammalian host during the blood-stage cycle. Anopheles mosquitoes depend on mammalian blood to lay eggs and to transmit malaria parasites. However, it remains understudied whether changes in host metabolism affect parasite transmission in mosquitoes. In this study, we discovered that Plasmodium infection significantly decreased the levels of the tryptophan metabolite, 5-hydroxytryptamine (5-HT), in both humans and mice. The reduction led to the decrease of 5-HT in mosquitoes. Oral supplementation of 5-HT to Anopheles stephensi enhanced its resistance to Plasmodium berghei infection by promoting the generation of mitochondrial reactive oxygen species. This effect was due to the accumulation of dysfunctional mitochondria caused by 5-HT-mediated inhibition of mitophagy. Elevating 5-HT levels in mouse serum significantly suppressed parasite infection in mosquitoes. In summary, our data highlight the critical role of metabolites in animal blood in determining the capacity of mosquitoes to control parasite infection. Author summary: Plasmodium infection causes symptoms ranging from fever, headache, convulsions to death that are associated with metabolic dysregulation in patients. Because Plasmodium is transmitted to mosquitoes through blood, it remains unclear how host metabolic changes affect parasite transmission in mosquitoes. In this study, we show that Plasmodium infection reduces the metabolite 5-HT in humans, mice and mosquitoes. Artificially increasing and decreasing 5-HT levels in mosquitoes inhibits and facilitates Plasmodium infection in mosquitoes, respectively. Mechanistic studies show that 5-HT inhibits mitophagy, leading to the accumulation of dysfunctional mitochondria, which exacerbates ROS generation. The increase in ROS, in turn, promotes parasite clearance. We also show that restoring the 5-HT levels in Plasmodium-infected mice to those of uninfected mice effectively suppresses parasite infection in mosquitoes. Collectively, these findings suggest the potential of correcting host metabolism to inhibit vector-borne parasite transmission. [ABSTRACT FROM AUTHOR]

Background: The interplay of OGG1, 8-Oxoguanine, and oxidative stress triggers the exaggerated release of cytokines during malaria, which worsens the outcome of the disease. We aimed to investigate the involvement of OGG1 in malaria and assess the effect of modulating its activity on the cytokine environment and anemia during P. berghei malaria in mice. Methods: Plasmodium berghei ANKA infection in ICR mice was used as a malaria model. OGG1 concentration and oxidative stress levels in P. berghei-infected mice and their control counterparts were assessed during malaria using enzyme-linked immunosorbent assay. OGG1 activity in malaria mice was modulated using treatment with TH5487 and O8-OGG1 inhibitors. The effects of modulating OGG1 activity using OGG1 inhibitors on cytokine release and anemia during P. berghei malaria infection were assessed by cytometric bead array and measurement of total normal red blood cell count respectively. Results: The plasma OGG1 level was significantly upregulated and positively correlated with parasitemia during P. berghei malaria in mice. Modulation of OGG1 ameliorated malaria severity by improving the total normal RBC count in TH5487 and O8-treated mice. Modulation of OGG1 with TH5487 caused significant reductions in serum levels of TNF-α, IFN-γ, IL-6, and IL-10. Similarly, OGG1 modulation activity using an O8-OGG1 inhibitor caused a significant reduction in serum levels of TNF-a, IL-2, IL-6, and IL-10. Conclusion: The findings indicate the involvement of OGG1 in the P. berghei malaria infection. OGG1 inhibition by TH5487 and O8-OGG1 inhibitors suppressed excessive cytokine release, and this may represent a novel therapeutic strategy for ameliorating the severity of malaria infection. [ABSTRACT FROM AUTHOR]

In both humans and mice, natural killer (NK) cells are important lymphocytes of the innate immune system. They are often considered pro-inflammatory effector cells but may also have a regulatory or pro-resolving function by switching their cytokine profile towards the production of anti-inflammatory cytokines, including interleukin-10 (IL-10) and transforming growth factor-p, and by killing pro-inflammatory immune cells. Here, the role of NK cells in the resolution of malaria lung pathology was studied. Malaria complications, such as malaria-associated acute respiratory distress syndrome (MA-ARDS), are often lethal despite the rapid and efficient killing of Plasmodium parasites with antimalarial drugs. Hence, studying the resolution and healing mechanisms involved in the recovery from these complications could be useful to develop adjunctive treatments. Treatment of Plasmodium berghei NK65-infected C57BL/6 mice with a combination of artesunate and chloroquine starting at the appearance of symptoms was used as a model to study the resolution of MA-ARDS. The role of NK cells was studied using anti-NK1.1 depletion antibodies and NK cell-deficient mice. Using both methods, NK cells were found to be dispensable in the development of MA-ARDS, as shown previously. In contrast, NK cells were crucial in the initiation of resolution upon antimalarial treatment, as survival was significantly decreased in the absence of NK cells. Considerably increased IL-10 expression by NK cells suggested an antiinflammatory and pro-resolving phenotype. Despite the increase in Il10 expression in the NK cells, inhibition of the IL-10/IL-10R axis using anti-IL10R antibodies had no effect on the resolution for MA-ARDS, suggesting that the pro-resolving effect of NK cells cannot solely be attributed to their IL-10 production. In conclusion, NK cells contribute to the resolution of experimental MA-ARDS. [ABSTRACT FROM AUTHOR]

Background/Objectives: We developed a multistage Plasmodium falciparum vaccine using a heterologous prime-boost immunization strategy. This involved priming with a highly attenuated, replication-competent vaccinia virus strain LC16m8Δ (m8Δ) and boosting with adeno-associated virus type 1 (AAV1). This approach demonstrated 100% efficacy in both protection and transmission-blocking in a murine model. In this study, we compared our LC16m8∆/AAV1 vaccine, which harbors a gene encoding Pfs25-PfCSP fusion protein, to RTS,S/AS01 (RTS,S) in terms of immune responses, protective efficacy, and transmission-blocking activity (TBA) in murine models. Methods: Mice were immunized following prime-boost vaccine regimens m8∆/AAV1 or RTS,S and challenged with transgenic Plasmodium berghei parasites. Immune responses were assessed via ELISA, and TB efficacy was evaluated using direct feeding assays. Results: m8∆/AAV1 provided complete protection (100%) in BALB/c mice and moderate (40%) protection in C57BL/6 mice, similar to RTS,S. Unlike RTS,S's narrow focus (repeat region), m8∆/AAV1 triggered antibodies for all PfCSP regions (N-terminus, repeat, and C-terminus) with balanced Th1/Th2 ratios. Regarding transmission blockade, serum from m8∆/AAV1-vaccinated BALB/c mice achieved substantial transmission-reducing activity (TRA = 83.02%) and TB activity (TBA = 38.98%)—attributes not observed with RTS,S. Furthermore, m8∆/AAV1 demonstrated durable TB efficacy (94.31% TRA and 63.79% TBA) 100 days post-immunization. Conclusions: These results highlight m8∆/AAV1′s dual action in preventing sporozoite invasion and onward transmission, a significant advantage over RTS,S. Consequently, m8∆/AAV1 represents an alternative and a promising vaccine candidate that can enhance malaria control and elimination strategies. [ABSTRACT FROM AUTHOR]

Objective: to determine the histopathological picture of the spleen of mice (Mus musculus Balb/C) infected with Plasmodium berghei which has been immunized with An. sundaicus salivary gland extract. Method: This type of research is a descriptive study with a quantitative approach. This research was conducted at the Histology Laboratory of the Faculty of Medicine, Andalas University, from March 2023 to September 2024. Result: research on the histopathological picture of the spleen of mice Mus musculus Balb / C infected with Plasmodium berghei showed the number of spleen cell megakaryocytes in the control group with an average of 2.63 per field of view, in the pellet extract group 2.23 per field of view and in the supernatant extract group 2.13 per field of view. Conclusion: the average megakaryocyte cells in the spleen were the highest in mice injected with the control group and the average megakaryocyte cells in the spleen were the lowest in mice injected with the supernatant extract group.

Abstract Background The spread of antimalarial drug resistance parasites is a major obstacle in eliminating malaria in endemic areas. This increases the urgency for developing novel antimalarial drugs with improved profiles to eliminate both sensitive and resistant parasites in populations. The invention of the drug candidates needs a model for sensitive and resistant parasites on a laboratory scale. Methods Repeated Incomplete Treatment (RIcT) method was followed in raising the rodent malaria parasite, Plasmodium berghei, resistant to sulfadoxine. Plasmodium berghei were exposed to an adequate therapeutic dose of sulfadoxine without finishing the treatment to let the parasite recover. Cycles of drug treatment and parasite recovery were repeated until phenotypic resistance appeared. Results After undergoing 3–4 cycles, phenotypic resistance was not yet found in mice treated with sulfadoxine. Nevertheless, the molecular biology of dhps gene (the target of sulfadoxine) was analyzed at the end of the RIcT cycle. There was no mutations found in the gene target. Interestingly, the appearance of gametocytes at the end of every cycle of drug treatment and parasite recovery was observed. These gametocytes later on would no longer extend their life in the RBC stage, unless mosquitoes bite the infected host. This phenomenon is similar to the case in human malaria infections treated with sulfadoxine-pyrimethamine (SP). Conclusions In this study, the antimalarial drug sulfadoxine induced gametocytogenesis in P. berghei, which could raise the risk factor for malaria transmission.

Objective: To evaluate the antimalarial activity of noscapine against Plasmodium falciparum 3D7 strain (Pf3D7), its clinical isolate (Pf140/SS), and Plasmodium berghei ANKA (PbA). Methods: Using ring-stage survival assay, phenotypic assessments, and SYBR-green-based fluorescence assay, the antimalarial activities of noscapine were assessed compared with dihydroartemisinin (DHA) in in vivo and in vitro studies. In addition, hemolysis and cytotoxicity tests were carried out to evaluate its safety. RT-PCR assay was also conducted to determine the effect of noscapine on papain-like cysteine protease Plasmodium falciparum falcipain-2 (PfFP-2). Results: The antimalarial efficacy of noscapine against Pf3D7 and Pf140/SS was comparable to DHA, with IC50 values of (7.68±0.88) and (5.57±0.74) nM/mL, respectively, and >95% inhibition of PbA infected rats. Noscapine also showed a safe profile, as evidenced by low hemolysis and cytotoxicity even at high concentrations. Moreover, PfFP-2 expression was significantly inhibited in both noscapine-treated Pf3D7 and Pf140/SS (P

Background & Aims: Malaria disease imposes a substantial global health burden, urging the search for effective treatments amid escalating drug resistance. This study investigates the antiplasmodial potential of Azanza garckeana root bark extract in Plasmodium berghei-infected mice and evaluates its impact on hematological indices. Materials & Methods: In this experimental research, we studied on six groups of 30 mice (Groups A to F) comprising of five mice per group. Group A was only given food and water with no inoculation and treatment, B was inoculated with Plasmodium berghei but no treatment was given, C was infected and treated with artemether while D E F were infected and treated with 100mg/kg, 200mg/kg and 300mg/kg of Azanza garckeana root-bark extract respectively for 4 days. Parasitemia levels, chemo-suppressive effect and hematological parameters were assessed on four different days following the start of the treatment. The findings were expressed as mean ± SEM. One-way analysis of variance was used to find the differences between the four groups, and p < 0.05 was considered statistically significant. Results: Phytochemical analysis revealed diverse bioactive compounds in the extract. The 4-day suppressive test demonstrated substantial antiplasmodial activity, with the 300 mg/kg dose achieving an 88.17% chemo-suppressive effect, comparable to Artemether's efficacy. Significant (p < 0.05) increases in hemoglobin, packed cell volume, red blood cells, white blood cells, and platelets were seen in hematological examinations in intervention groups, especially with the 300 mg/kg dosage. Conclusions: Azanza garckeana root-bark extract exhibited potent antiplasmodial effects, possibly mediated by identified phytochemicals. The dose-dependent chemosuppression and modulation of hematological parameters underscore its potential as an alternative antimalarial therapy in mammals.

Introduction: In recent years, due to the spread of drug resistance in the Plasmodium parasite and the shortcomings of anti-malarial drugs, the use of liposome-based nanomedicines in treating malaria has garnered significant attention. This research studied the effect of liposomes containing the antimalarial compound Eosin B on the Plasmodium berghei parasite in murine malaria.Methods: Liposomes containing Eosin B were prepared using cholesterol in the presence of Eosin B via solvent evaporation. Eosin B, Eosin B liposome, drug solvent, drug-free liposomes, and control drugs were administered to infected mice. The percentage of parasite inhibitory activity was evaluated using Peter’s test. In the in vitro model, the Plasmodium falciparum parasite was first cultured, and the survival percentage of the parasite in each group was investigated using the parasite lactate dehydrogenase test.Results: In each test group, the percentage of parasitemia under the effect of the Eosin B liposome was lower than that of Eosin B alone, and the inhibition percentage was significantly higher (P

Abstract Background Malaria continues to wreak havoc on the well-being of the community. Resistant parasites are jeopardizing the treatment. This is a wake-up call for better medications. Folk plants are the key starting point for antimalarial drug discovery. After crushing and mixing the leaves of Coriandrum sativum with water, one cup of tea is drunk daily for a duration of three to five days as a remedy for malaria by local folks in Ethiopia. Additionally, in vitro experiments conducted on the plant leaf extract elsewhere have also demonstrated the plant’s malaria parasite inhibitory effect. There has been no pharmacologic research to assert this endowment in animals, though. This experiment was aimed at evaluating the antimalarial efficacy of C. sativum in Plasmodium berghei infected mice. Methods The plant's leaf was extracted using maceration with distilled water. The extract was examined for potential acute toxicity. An evaluation of secondary phytoconstituents was done. Standard antimalarial screening models (prophylactic, chemosuppressive, curative tests) were utilized to assess the antiplasmodial effect. In each test, thirty mice were organized into groups of five. To the three categories, the test substance was given at doses of 100, 200 and 400 mg/kg/day before or after the commencement of P. berghei infection. Positive and negative control mice were provided Chloroquine and distilled water, respectively. Rectal temperature, parasitemia, body weight, survival time and packed cell volume were ultimately assessed. Analysis of the data was performed using Statistical Package for Social Sciences. Results No toxicity was manifested in mice. The extract demonstrated a significant inhibition of parasitemia (p

Abstract Background Malaria, a global health concern, is caused by parasites of the Plasmodium genus, which undergo gametogenesis in the midgut of mosquitoes after ingestion of an infected blood meal. The resulting male and female gametes fuse to form a zygote, which differentiates into a motile ookinete. After traversing the midgut epithelium, the ookinete differentiates into an oocyst on the basal side of the epithelium. Methods Membrane proteins with increased gene expression levels from the gamete to oocyst stages in P. berghei were investigated utilizing PlasmoDB, the functional genomic database for Plasmodium spp. Based on this analysis, we selected the 184-kDa membrane protein, Pb184, for further study. The expression of Pb184 was further confirmed through immunofluorescence staining, following which we examined whether Pb184 is involved in fertilization using antibodies targeting the C-terminal region of Pb184 and biotin-labeled C-terminal region peptides of Pb184. Results Pb184 is expressed on the surface of male and female gametes. The antibody inhibited zygote and ookinete formation in vitro. When mosquitoes were fed on parasite-infected blood containing the antibody, oocyst formation decreased on the second day after feeding. Synthesized biotin-labeled peptides matching the C-terminal region of Pb184 bound to the female gamete and the residual body of male gametes, and inhibited differentiation into ookinetes in the in vitro culture system. Conclusions These results may be useful for the further studying the fertilization mechanism of Plasmodium protozoa. There is also the potential for their application as future tools to prevent malaria transmission. Graphical Abstract

Background: Severe malaria can cause respiratory symptoms, which may lead to malaria-acute lung injury (MA-ALI) due to inflammation and damage to the blood-gas barrier. Patients with severe malaria also often present thrombocytopenia, and the use of acetylsalicylic acid (ASA), a commonly used non-steroidal anti-inflammatory drug with immunomodulatory and antiplatelet effects, may pose a risk in regions where malaria is endemic. Thus, this study aimed to investigate the systemic impact of ASA and dihydroartemisinin (DHA) on ALI induced in mice by Plasmodium berghei NK65 (PbNK65). Methods: C57BL/6 mice were randomly divided into control (C) and PbNK65 infected groups and were inoculated with uninfected or 104 infected erythrocytes, respectively. Then, the animals were treated with DHA (3 mg/kg) or vehicle (DMSO) at the 8-day post-infection (dpi) for 7 days and with ASA (100 mg/kg, single dose), and analyses were performed at 9 or 15 dpi. Lung mechanics were performed, and lungs were collected for oedema evaluation and histological analyses. Results: PbNK65 infection led to lung oedema, as well as increased lung static elastance (Est, L), resistive (ΔP1, L) and viscoelastic (ΔP2, L) pressures, percentage of mononuclear cells, inflammatory infiltrate, hemorrhage, alveolar oedema, and alveolar thickening septum at 9 dpi. Mice that received DHA or DHA + ASA had an increase in Est, L, and CD36 expression on inflammatory monocytes and higher protein content on bronchoalveolar fluid (BALF). However, only the DHA-treated group presented a percentage of inflammatory monocytes similar to the control group and a decrease in ΔP1, L and ΔP2, L compared to Pb + DMSO. Also, combined treatment with DHA + ASA led to an impairment in diffuse alveolar damage score and lung function at 9 dpi. Conclusions: Therapy with ASA maintained lung morpho-functional impairment triggered by PbNK65 infection, leading to a large influx of inflammatory monocytes to the lung tissue. Based on its deleterious effects in experimental MA-ALI, ASA administration or its treatment maintenance might be carefully reconsidered and further investigated in human malaria cases. [ABSTRACT FROM AUTHOR]

Background: Transmission-blocking vaccines (TBVs) can effectively prevent the community's spread of malaria by targeting the antigens of mosquito sexual stage parasites. At present, only a few candidate antigens have demonstrated transmission-blocking activity (TBA) potential in P. vivax. Quiescin-sulfhydryl oxidase (QSOX) is a sexual stage protein in the rodent malaria parasite Plasmodium berghei and is associated with a critical role in protein folding by introducing disulfides into unfolded reduced proteins. Here, we reported the immunogenicity and transmission-blocking potency of the PvQSOX in P. vivax. Methods and findings: The full-length recombinant PvQSOX protein (rPvQSOX) was expressed in the Escherichia coli expression system. The anti-rPvQSOX antibodies were generated following immunization with the rPvQSOX in rabbits. A parasite integration of the pvqsox gene into the P. berghei pbqsox gene knockout genome was developed to express full-length PvQSOX protein in P. berghei (Pv-Tr-PbQSOX). In western blot, the anti-rPvQSOX antibodies recognized the native PvQSOX protein expressed in transgenic P. berghei gametocyte and ookinete. In indirect immunofluorescence assays, the fluorescence signal was detected in the sexual stages, including gametocyte, gamete, zygote, and ookinete. Anti-rPvQSOX IgGs obviously inhibited the ookinetes and oocysts development both in vivo and in vitro using transgenic parasites. Direct membrane feeding assays of anti-rPvQSOX antibodies were conducted using four field P. vivax isolates (named isolates #1-4) in Thailand. Oocyst density in mosquitoes was significantly reduced by 32.00, 85.96, 43.52, and 66.03% with rabbit anti-rPvQSOX antibodies, respectively. The antirPvQSOX antibodies also showed a modest reduction of infection prevalence by 15, 15, 20, and 22.22%, respectively, as compared to the control, while the effect was insignificant. The variation in the DMFA results may be unrelated to the genetic polymorphisms. Compared to the P.vivax Salvador (Sal) I strain sequences, the pvqsox in isolate #1 showed no amino acid substitution, whereas isolates #2, #3, and #4 all had the M361I substitution. Conclusions: Our results suggest that PvQSOX could serve as a potential P. vivax TBVs candidate, which warrants further evaluation and optimization. [ABSTRACT FROM AUTHOR]

Background: The spread of antimalarial drug resistance parasites is a major obstacle in eliminating malaria in endemic areas. This increases the urgency for developing novel antimalarial drugs with improved profiles to eliminate both sensitive and resistant parasites in populations. The invention of the drug candidates needs a model for sensitive and resistant parasites on a laboratory scale. Methods: Repeated Incomplete Treatment (RIcT) method was followed in raising the rodent malaria parasite, Plasmodium berghei, resistant to sulfadoxine. Plasmodium berghei were exposed to an adequate therapeutic dose of sulfadoxine without finishing the treatment to let the parasite recover. Cycles of drug treatment and parasite recovery were repeated until phenotypic resistance appeared. Results: After undergoing 3–4 cycles, phenotypic resistance was not yet found in mice treated with sulfadoxine. Nevertheless, the molecular biology of dhps gene (the target of sulfadoxine) was analyzed at the end of the RIcT cycle. There was no mutations found in the gene target. Interestingly, the appearance of gametocytes at the end of every cycle of drug treatment and parasite recovery was observed. These gametocytes later on would no longer extend their life in the RBC stage, unless mosquitoes bite the infected host. This phenomenon is similar to the case in human malaria infections treated with sulfadoxine-pyrimethamine (SP). Conclusions: In this study, the antimalarial drug sulfadoxine induced gametocytogenesis in P. berghei, which could raise the risk factor for malaria transmission. [ABSTRACT FROM AUTHOR]

Bovine lactoferrin (bLF) is a 77 kDa glycoprotein that is abundant in bovine breast milk and exerts various bioactive functions, including antibacterial and antiviral functions. Few studies have explored bLF activity against parasites. We found that bLF affects hemozoin synthesis by binding to heme, inhibiting heme iron polymerization necessary for Plasmodium berghei ANKA survival in infected erythrocytes, and also binds to hemozoin, causing it to disassemble. In a challenge test, bLF administration inhibited the growth of murine malaria parasites compared to untreated group growth. To determine whether the iron content of bLF affects the inhibition of malaria growth, we tested bLFs containing different amounts of iron (apo-bLF, native-bLF, and holo-bLF), but found no significant difference in their effects. This indicated that the active sites were located within the bLFs themselves. Further studies showed that the C-lobe domain of bLF can inhibit hemozoin formation and the growth of P. berghei ANKA. Evaluation of pepsin degradation products of the C-lobe identified a 47-amino-acid section, C-1, as the smallest effective region that could inhibit hemozoin formation. This study highlights bLF's potential as a novel therapeutic agent against malaria, underscoring the importance of its non-iron-dependent bioactive sites in combating parasite growth. [ABSTRACT FROM AUTHOR]

Background: Despite thewidely reported potentials of n-Hexadecanoic acid (HA) as a bioactive, its multi-stage antiplasmodial activity and toxicity profiles remain largely unknown. Methodology: Thus, this study uses a combination of in silico approaches and in vivo studies to assess the inhibitory activities of HA at different stages of the Plasmodium lifecycle, antiplasmodial performance, and toxicity profiles. The HA was retrieved from the PubChem database, while antiplasmodial target proteins from different stages of the Plasmodium falciparum life cycle were collated from the Protein Databank (PDB). Molecular Docking and Visualization were conducted between the compound and target proteins using AutoVina PyRx software and Biovia Discovery Studio, respectively. Also, the AdmetLab 3.0 algorithm was used to predict the absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) profiles of HA. Based on a 4-day suppressive test, the antiplasmodial activity against the Plasmodium berghei ANKA strain in mice was evaluated. Furthermore, subacute toxicity and micronucleus assays were used for further toxicity assessment. Results: The molecular docking analysis indicates multi-stage, multi-target potentials of HA with favourable ligand-receptor complexes across the four Plasmodium falciparum stages. Meanwhile, the mice administered with 100 mg/kg, 50 mg/kg, and 10 mg/kg of HA demonstrated considerable chemosuppression in a dose-dependent manner of 89.74%, 83.80%, and 71.58% percentage chemosuppression, respectively, at p < 0.05. The ADMET prediction, histopathological tests, andmicronucleus assays show that HA is safer at a lower dose. Conclusion: This study showed that n-Hexadecanoic acid is a potential drug candidate for malaria. Hence, it is recommended for further molecular and biochemical investigations. [ABSTRACT FROM AUTHOR]

The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers selfproperties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanizationbased immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only latestage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes frommelanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization. [ABSTRACT FROM AUTHOR]

Background: Malaria remains a significant global health burden, with drug resistance posing a major challenge to its control. The emergence of resistance to antimalarial drugs represents a critical issue in malaria management, as it heightens the likelihood of morbidity and mortality associated with the disease. There is an urgent requirement for a novel candidate drug with a distinct mechanism of action. Aim: In light of the ongoing challenges in malaria management, particularly the emergence of drug resistance, this study aimed to investigate the efficacy of a novel combination therapy of borrelidin and fumagilin against Plasmodium berghei infection on Swiss Webster mice. The findings of this study could contribute to developing new and effective antimalarial treatments. Methods: This study employed a unique approach, using Swiss Webster mice aged 6–8 weeks and dividing them into five groups, each with five mice. The therapeutic efficacy of the combination treatment was evaluated through a comprehensive assessment of parasitemia levels, survival rates, and histological changes in the liver and spleen. This rigorous methodology ensures the reliability and validity of our findings. Results: The combination of borrelidin and fumagilin led to the lowest parasitemia at 5%, contrasting with the control group reaching 15%. Moreover, the combination group exhibited the highest inhibition rate of 69.6% on day nine postinfection. Histopathological alterations were limited to sinusoid dilation, hepatocyte ballooning, and the presence of hemozoin. Conclusion: These findings suggest that the combination of borrelidin and fumagilin holds promise as a potential antimalarial therapy. [ABSTRACT FROM AUTHOR]

[Display omitted] • A new Plasmodium parasite was discovered in the horseshoe bat Rhinolophus landeri. • We found 100% prevalence of Plasmodium cyclopsi infections in Doryrhina cyclops bats in Sierra Leone. • We provide the first known analysis of complete mitochondrial genomes of bat Plasmodium spp. • There is a close relationship between bat Plasmodium and the rodent taxon Plasmodium berghei. • Distribution of Plasmodium spp. of bats might be limited to Africa. Malaria remains the most important arthropod-borne infectious disease globally. The causative agent, Plasmodium, is a unicellular eukaryote that develops inside red blood cells. Identifying new Plasmodium parasite species that infect mammalian hosts can shed light on the complex evolution and diversity of malaria parasites. Bats feature a high diversity of microorganisms including seven separate genera of malarial parasites. Three species of Plasmodium have been reported so far, for which scarce reports exist. Here we present data from an investigation of Plasmodium infections in bats in the western Guinean lowland forest in Sierra Leone. We discovered a new Plasmodium parasite in the horseshoe bat Rhinolophus landeri. Plasmodium cyclopsi infections in a member of leaf-nosed bats, Doryrhina cyclops, exhibited a high prevalence of 100%. Phylogenetic analysis of complete mitochondrial genomes and nine nuclear markers recovered a close relationship between P. cyclopsi and the new Plasmodium parasite with the rodent species Plasmodium berghei , a widely used in vivo model to study malaria in humans. The data suggests that the "rodent/bat" Plasmodium (Vinckeia) clade represents a diverse group of malarial parasites that would likely expand with a systematic sampling of small mammals in tropical Africa. Identifying the bat Plasmodium repertoire is central to our understanding of the evolution of Plasmodium parasites in mammals. [ABSTRACT FROM AUTHOR]

Although many drugs have been discovered to treat malaria infection, many of them face resistance from the host's body with long-term use. Therefore, this study aimed to evaluate the activity of betalains (from Beta vulgaris) and chloroquine (a reference drug) against brain oxidative stress induced by Plasmodium berghei in male mice. Two protocols were applied in this study: the therapeutic and prophylactic protocols. The results of the therapeutic protocol revealed a significant decrease in the level of parasitemia caused by P. berghei. Additionally, the histopathological changes in various brain regions were markedly improved after treatment with betalains. Regarding the prophylactic protocol, betalains were able to protect the brain tissues from oxidative stress, inflammation, and disrupted neurotransmitters expected to occur as a result of infection by P. berghei. This was demonstrated by modulating the activities of brain antioxidants (SOD and GSH), inflammatory cytokines (IL-6, IL-10, IL-12, TNF-α, and INF-γ), and neurotransmitters (serotonin, epinephrine, and norepinephrine). This study has proven that using betalains as a treatment or as a preventive has a vital and effective role in confronting the brain histopathological, oxidative stress, and inflammatory changes induced by P. berghei infection. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the antimalarial activity of noscapine against Plasmodium falciparum 3D7 strain (Pf3D7), its clinical isolate (Pf140/SS), and Plasmodium berghei ANKA (PbA). Methods: Using ring-stage survival assay, phenotypic assessments, and SYBR-green-based fluorescence assay, the antimalarial activities of noscapine were assessed compared with dihydroartemisinin (DHA) in in vivo and in vitro studies. In addition, hemolysis and cytotoxicity tests were carried out to evaluate its safety. RT-PCR assay was also conducted to determine the effect of noscapine on papain-like cysteine protease Plasmodium falciparum falcipain-2 (PfFP-2). Results: The antimalarial efficacy of noscapine against Pf3D7 and Pf140/SS was comparable to DHA, with IC50 values of (7.68±0.88) and (5.57±0.74) nM/mL, respectively, and >95% inhibition of PbA infected rats. Noscapine also showed a safe profile, as evidenced by low hemolysis and cytotoxicity even at high concentrations. Moreover, PfFP-2 expression was significantly inhibited in both noscapine-treated Pf3D7 and Pf140/SS (P <0.01). Conclusions: Noscapine has antimalarial properties comparable to standard antimalarial DHA with better safety profiles, which may be further explored as a therapeutic candidate for the treatment of malaria. [ABSTRACT FROM AUTHOR]

Plasmodium falciparum causes severe malaria and assembles a protein translocon (PTEX) complex at the parasitophorous vacuole membrane (PVM) of infected erythrocytes, through which several hundred proteins are exported to facilitate growth. The preceding liver stage of infection involves growth in a hepatocyte-derived PVM; however, the importance of protein export during P. falciparum liver infection remains unexplored. Here, we use the FlpL/FRT system to conditionally excise genes in P. falciparum sporozoites for functional liver-stage studies. Disruption of PTEX members ptex150 and exp2 did not affect sporozoite development in mosquitoes or infectivity for hepatocytes but attenuated liver-stage growth in humanized mice. While PTEX150 deficiency reduced fitness on day 6 postinfection by 40%, EXP2 deficiency caused 100% loss of liver parasites, demonstrating that PTEX components are required for growth in hepatocytes to differing degrees. To characterize PTEX loss-of-function mutations, we localized four liver-stage Plasmodium export element (PEXEL) proteins. P. falciparum liver specific protein 2 (LISP2), liver-stage antigen 3 (LSA3), circumsporozoite protein (CSP), and a Plasmodium berghei LISP2 reporter all localized to the periphery of P. falciparum liver stages but were not exported beyond the PVM. Expression of LISP2 and CSP but not LSA3 was reduced in ptex150-FRT and exp2-FRT liver stages, suggesting that expression of some PEXEL proteins is affected directly or indirectly by PTEX disruption. These results show that PTEX150 and EXP2 are important for P. falciparum development in hepatocytes and emphasize the emerging complexity of PEXEL protein trafficking. [ABSTRACT FROM AUTHOR]

Background: Malaria continues to wreak havoc on the well-being of the community. Resistant parasites are jeopardizing the treatment. This is a wake-up call for better medications. Folk plants are the key starting point for antimalarial drug discovery. After crushing and mixing the leaves of Coriandrum sativum with water, one cup of tea is drunk daily for a duration of three to five days as a remedy for malaria by local folks in Ethiopia. Additionally, in vitro experiments conducted on the plant leaf extract elsewhere have also demonstrated the plant's malaria parasite inhibitory effect. There has been no pharmacologic research to assert this endowment in animals, though. This experiment was aimed at evaluating the antimalarial efficacy of C. sativum in Plasmodium berghei infected mice. Methods: The plant's leaf was extracted using maceration with distilled water. The extract was examined for potential acute toxicity. An evaluation of secondary phytoconstituents was done. Standard antimalarial screening models (prophylactic, chemosuppressive, curative tests) were utilized to assess the antiplasmodial effect. In each test, thirty mice were organized into groups of five. To the three categories, the test substance was given at doses of 100, 200 and 400 mg/kg/day before or after the commencement of P. berghei infection. Positive and negative control mice were provided Chloroquine and distilled water, respectively. Rectal temperature, parasitemia, body weight, survival time and packed cell volume were ultimately assessed. Analysis of the data was performed using Statistical Package for Social Sciences. Results: No toxicity was manifested in mice. The extract demonstrated a significant inhibition of parasitemia (p < 0.05) in all the models. The inhibition of parasite load was highest with the upper dose in the suppressive test (82.74%) followed by the curative procedure (78.49%). Likewise, inhibition of hypothermia, weight loss hampering, improved survival and protection against hemolysis were elicited by the extract. Conclusions: The results of our experimental study revealed that the aqueous crude leaf extract of C. sativum exhibits significant antimalarial efficacy in multiple in vivo models involving mice infected with P. berghei. Given this promising therapeutic attribute, in depth investigation on the plant is recommended. [ABSTRACT FROM AUTHOR]

Prosopis africana, a traditional medicinal plant for malaria treatment, requires further scientific evidence. This study aimed to assess the antimalarial potential of Prosopis africana stem bark fractions in Plasmodium berghei-infected mice and identify the responsible compounds. The aqueous extract underwent partitioning with n-hexane, ethyl acetate, and methanol, followed by antimalarial evaluation. F5 was the most potent fraction based on its antimalarial potential. The study pooled 36 chromatographic separation sub-fractions into five using TLC analysis Rf values. In Plasmodium berghei-infected mice, fraction F5 at 6.25, 12.5, 25, and 50 mg/kg body weight (bw) was tested for antimalarial activity. Compound analysis of F5 utilized ultraperformance liquid chromatography-quadrupolar mass spectrometry. Statistical analysis employed Analysis of Variance and Duncan's multiple range tests (p < 0.05). The methanol, n-hexane, and ethylacetate extracts displayed suppression of P. berghei by 90%, 72.2%, and 73.6%, respectively. Methanol, n-hexane, and ethylacetate extracts suppressed P. berghei by 90%, 72.2%, and 73.6% respectively. F5 showed significantly higher chemo-suppression (67.21%) than F1 (63.94%), F2 (6.99%), F3 (2.81%), and F4 (37.3%). At 12.5 mg/kg bw, F5 suppressed P. berghei by 91.09%, surpassing chloroquine (87.79%), and restored altered hematological indices and CD4+ counts. Compounds identified in F5 included caffeate, linalool, terpinene-4-ol, quinovic acid, quercetin, prosopinine, catechin, and apigeninin. In conclusion, the fraction F5 of P. africana stem bark exhibited higher antimalarial activity than chloroquine and the identified compound might likely contribute to its activity. [ABSTRACT FROM AUTHOR]

Introduction: In recent years, due to the spread of drug resistance in the Plasmodium parasite and the shortcomings of anti-malarial drugs, the use of liposome-based nanomedicines in treating malaria has garnered significant attention. This research studied the effect of liposomes containing the antimalarial compound Eosin B on the Plasmodium berghei parasite in murine malaria. Methods: Liposomes containing Eosin B were prepared using cholesterol in the presence of Eosin B via solvent evaporation. Eosin B, Eosin B liposome, drug solvent, drug-free liposomes, and control drugs were administered to infected mice. The percentage of parasite inhibitory activity was evaluated using Peter’s test. In the in vitro model, the Plasmodium falciparum parasite was first cultured, and the survival percentage of the parasite in each group was investigated using the parasite lactate dehydrogenase test. Results: In each test group, the percentage of parasitemia under the effect of the Eosin B liposome was lower than that of Eosin B alone, and the inhibition percentage was significantly higher (P<0.05). Also, the survival percentage of the parasite in the in vitro model was lower in each group under the effect of the Eosin B liposome compared to Eosin B alone. Conclusion: Liposomes containing Eosin B can increase efficacy in the mouse malaria model and reduce the drug’s dose. Considering the reduction of drug dosage in treating malaria by the Eosin B liposome, using this liposome drug might be beneficial. [ABSTRACT FROM AUTHOR]

Parasite-derived new permeation pathways (NPPs) expressed at the red blood cell (RBC) membrane enable Plasmodium parasites to take up nutrients from the plasma to facilitate their survival. Thus, NPPs represent a potential novel therapeutic target for malaria. The putative channel component of the NPP in the human malaria parasite P. falciparum is encoded by mutually exclusively expressed clag3.1/3.2 genes. Complicating the study of the essentiality of these genes to the NPP is the addition of three clag paralogs whose contribution to the P. falciparum channel is uncertain. Rodent malaria P. berghei contains only two clag genes, and thus studies of P. berghei clag genes could significantly aid in dissecting their overall contribution to NPP activity. Previous methods for determining NPP activity in a rodent model have utilised flux-based assays of radioisotope-labelled substrates or patch clamping. This study aimed to ratify a streamlined haemolysis assay capable of assessing the functionality of P. berghei NPPs. Several isotonic lysis solutions were tested for their ability to preferentially lyse infected RBCs (iRBCs), leaving uninfected RBCs (uRBCs) intact. The osmotic lysis assay was optimised and validated in the presence of NPP inhibitors to demonstrate the uptake of the lysis solution via the NPPs. Guanidinium chloride proved to be the most efficient reagent to use in an osmotic lysis assay to establish NPP functionality. Furthermore, following treatment with guanidinium chloride, ring-stage parasites could develop into trophozoites and schizonts, potentially enabling use of guanidinium chloride for parasite synchronisation. This haemolysis assay will be useful for further investigation of NPPs in P. berghei and could assist in validating its protein constituents. [ABSTRACT FROM AUTHOR]

Background and Aim: Brain malaria, which results from Plasmodiumfalciparum infection, is responsible for substantial fatalities and health issues. These processes, including cytoadherence, rosetting, and sequestration, induce an immune response, hypoxia, brain microvascular obstruction, disruption of the blood-brain barrier, and cell death. Parasitemia level can reveal the presence of infection and its association with apoptosis-related genes. Neem (Azadirachta indica) leaves with antimalarial properties could replace ineffective Indonesian malaria medications. This study was designed to evaluate the impact of neem leaf extract on cerebral malaria-induced parasitemia and neuron cell apoptosis in mice through an in vivo approach. Materials and Methods: 13-16 weeks old C57BL mice received infection by Plasmodium berghei strain ANKA. Parasitemia was estimated daily from the mice's tail blood. 8 mg, 12 mg, and 16 mg of a 96% ethanolic neem leaf extract were orally given for 6 days. Healthy, positive, and negative controls were included for treatment comparisons. On the 7th day, brain tissue was analyzed for (p > 0.05) gene expression. Through immunohistochemistry, both cell apoptosis in neurons expressing caspase-3 within a brain sample and the degree of parasitemia in a blood smear were assessed. The Pearson correlation test and one-way analysis of variance were employed to analyze the data. Results: Neem leaf extract reduces parasitemia and neuron cell apoptosis at multiple dosages (p < 0.000). Apoptosis in brain neurons and parasitemia show a strong positive correlation (r = +0.939). Neem leaf extract at doses of 12 and 16 mg was the most effective in reducing parasitemia levels and causing cell death. Conclusions: Neem leaf therapy significantly reduced the degree of parasitemia and cell apoptosis in C57BL mice compared with the control group without treatment (p = 0.05). This shows that neem leaves have the potential to be a candidate drug for malaria. [ABSTRACT FROM AUTHOR]

BACKGROUND: Salvia officinalis (Common Sage) plant, is used as herbal medicine. The study was aimed at investigating the antimalarial potential and liver function profiles of the Methanol Extract of Salvia officinalis. METHODS: Mice infected with Plasmodium berghei were treated (p.o) with the extract in the curative, suppressive, and prophylactic antimalarial models at doses of 250mg/kg, 500mg/kg, and 1000mg/kg. The positive control drug used was artemether/ lumefantrine (7mg/kg A/L) while the negative control was 10mk/kg of Tween 80. RESULTS: The curative stage showed a significant (p < 0.001) dose-dependent antiplasmodial effect (of the methanol extract of S. officinalis leaf) compared with the negative control (Group 1). At doses of 250, 500, and 1000mg/kg, the Salvia officinalis extract produced parasite suppression of 37.13%, 57.18% and 66.80% respectively. While the positive control group produced parasite percentage suppression of 74.38%. There was a significant chemosuppressive effect (p < 0.001) at all doses of the methanol extract of Salvia officinalis leaf.The leaf extracts demonstrated a prophylactic significant (p < 0.001) activity. There was no significant effect (p > 0.05) on packed cell volume at doses of 250 and 500mg/kg while 1000mg/kg body weight showed a significant (p < 0.05) effect. There was a reduction in the level of activity of the enzymes and other parameters in the liver function tests with an increase in the dosage of the leaf extract. CONCLUSION: The methanol extract of Salvia officinalis possesses in vivo antiplasmodial activities and could be a lead plant in the development of antiplasmodial agents. [ABSTRACT FROM AUTHOR]

Introduction: Emergence of drug resistant strains of Plasmodium species has necessitated the search for novel antimalarials with unique mechanisms of action. Synthesis of hybrid compounds has been one approach to tackling this challenge. In this study, the synthesis of artesunate-ellagic acid hybrid compound (EA31) from ellagic acid and artesunate and its evaluation for antimalarial and antioxidant activities using in vitro and in vivo models were carried out. Method: EA31 was synthesized from artesunate and ellagic acid. The activities of the hybrid compound against Plasmodium falciparum W2 and P. berghei NK65 were evaluated, and its antioxidant activities were also determined. Results: The results revealed that EA31 was more active against P. falciparum W2 (chloroquine resistant) clone and less cytotoxic to buffalo green monkey kidney cell line compared to artesunate. EA31 was also active against Plasmodium berghei NK65 in vivo. The results revealed inhibition of β-hematin formation as one of the mechanisms of action of EA31. EA31 also exhibited antioxidant activities. Conclusion: The results revealed that EA31 may exert dual action of killing malaria parasite and mopping the reactive oxygen species that mediate the secondary complications of malaria. [ABSTRACT FROM AUTHOR]

The Aurora family of kinases orchestrates chromosome segregation and cytokinesis during cell division, with precise spatiotemporal regulation of its catalytic activities by distinct protein scaffolds. Plasmodium spp., the causative agents of malaria, are unicellular eukaryotes with three unique and highly divergent aurora-related kinases (ARK1-3) that are essential for asexual cellular proliferation but lack most canonical scaffolds/activators. Here we investigate the role of ARK2 during sexual proliferation of the rodent malaria Plasmodium berghei, using a combination of super-resolution microscopy, mass spectrometry, and live-cell fluorescence imaging. We find that ARK2 is primarily located at spindle microtubules in the vicinity of kinetochores during both mitosis and meiosis. Interactomic and co-localisation studies reveal several putative ARK2-associated interactors including the microtubule-interacting protein EB1, together with MISFIT and Myosin-K, but no conserved eukaryotic scaffold proteins. Gene function studies indicate that ARK2 and EB1 are complementary in driving endomitotic division and thereby parasite transmission through the mosquito. This discovery underlines the flexibility of molecular networks to rewire and drive unconventional mechanisms of chromosome segregation in the malaria parasite.

The mainstay for malaria treatment is drug, but currently, the treatment is failing and making malaria infection worrisome. The alkaloid extract of the plant Phyllanthus amarus has been demonstrated to possess antimalarial activity. This present study evaluated the changes in semen quality, hormone profile and gonadal histomorphometric features in Berghei-induced mice treated with alkaloid extract of Phyllanthus amarus. Thirty-six BALB/c mice were divided into six groups (n=6 per group). The first two groups were positive (not infected, not treated) and negative (infected, but not treated) control mice, while the remaining groups (Groups 3–6) were the experimental mice infected with Plasmodium berghei (NK 65) malaria parasite and respectively treated with standard drug Lonart® DS (20 mg/kg) with 75 mg/kg, 150 mg/kg and 225 mg/kg of the Phyllanthus amarus alkaloid extract (PAAE) for 4 days. Then, 10 days later, the animals were euthanized and blood samples were collected for biochemical investigation. Testes tissues were harvested for histological assessment and semen samples were collected for analysis using standard procedures and documented methods. Data from our study revealed that Plasmodium berghei malaria-induced infection significantly (p

Background: Studying and discovering the molecular mechanism of Plasmodium sexual development is crucial for the development of transmission blocking drugs and malaria eradication. The aim of this study was to investigate the feasibility of using phosphatase inhibitors as a tool for screening proteins essential for Plasmodium sexual development and to discover proteins affecting the sexual development of malaria parasites. Methods: Differences in protein phosphorylation among Plasmodium gametocytes incubated with BVT-948 under in vitro ookinete culture conditions were evaluated using phosphoproteomic methods. Gene Ontology (GO) analysis was performed to predict the mechanism by which BVT-948 affected gametocyte–ookinete conversion. The functions of 8 putative proteins involved in Plasmodium berghei sexual development were evaluated. Bioinformatic analysis was used to evaluate the possible mechanism of PBANKA_0100800 in gametogenesis and subsequent sexual development. Results: The phosphorylation levels of 265 proteins decreased while those of 67 increased after treatment with BVT-948. Seven of the 8 genes selected for phenotype screening play roles in P. berghei sexual development, and 4 of these were associated with gametocytogenesis. PBANKA_0100800 plays essential roles in gametocyte–ookinete conversion and transmission to mosquitoes. Conclusions: Seven proteins identified by screening affect P. berghei sexual development, suggesting that phosphatase inhibitors can be used for functional protein screening.

Background: The interplay of OGG1, 8-Oxoguanine, and oxidative stress triggers the exaggerated release of cytokines during malaria, which worsens the outcome of the disease. We aimed to investigate the involvement of OGG1 in malaria and assess the effect of modulating its activity on the cytokine environment and anemia during P. berghei malaria in mice. Methods: Plasmodium berghei ANKA infection in ICR mice was used as a malaria model. OGG1 concentration and oxidative stress levels in P. berghei-infected mice and their control counterparts were assessed during malaria using enzyme-linked immunosorbent assay. OGG1 activity in malaria mice was modulated using treatment with TH5487 and O8-OGG1 inhibitors. The effects of modulating OGG1 activity using OGG1 inhibitors on cytokine release and anemia during P. berghei malaria infection were assessed by cytometric bead array and measurement of total normal red blood cell count respectively. Results: The plasma OGG1 level was significantly upregulated and positively correlated with parasitemia during P. berghei malaria in mice. Modulation of OGG1 ameliorated malaria severity by improving the total normal RBC count in TH5487 and O8-treated mice. Modulation of OGG1 with TH5487 caused significant reductions in serum levels of TNF-α, IFN-γ, IL-6, and IL-10. Similarly, OGG1 modulation activity using an O8-OGG1 inhibitor caused a significant reduction in serum levels of TNF-α, IL-2, IL-6, and IL-10. Conclusion: The findings indicate the involvement of OGG1 in the P. berghei malaria infection. OGG1 inhibition by TH5487 and O8-OGG1 inhibitors suppressed excessive cytokine release, and this may represent a novel therapeutic strategy for ameliorating the severity of malaria infection.

Chemical genetic approaches have had a transformative impact on discovery of drug targets for malaria but have primarily been used for parasite targets. To identify human pathways required for intrahepatic development of parasite, we implemented multiplex cytological profiling of malaria infected hepatocytes treated with liver stage active compounds. Some compounds, including MMV1088447 and MMV1346624, exhibited profiles similar to cells treated with nuclear hormone receptor (NHR) agonist/antagonists. siRNAs targeting human NHRs, or their signaling partners identified eight genes that were critical for Plasmodium berghei infection. Knockdown of NR1D2, a host NHR, significantly impaired parasite growth by downregulation of host lipid metabolism. Importantly, treatment with MMV1088447 and MMV1346624 but not other antimalarials, phenocopied the lipid metabolism defect of NR1D2 knockdown. Our data underlines the use of high-content imaging for host-cellular pathway deconvolution, highlights host lipid metabolism as a drug-able human pathway and provides new chemical biology tools for studying host-parasite interactions.

Background: In the midst of transient conquests by medications against malaria, Plasmodium parasites are the foremost public health problem globally. The emergence of drug-resistant parasites has worsened the condition, emphasizing the need for effective and inexpensive antimalarial. In line with this necessity, the study aimed at evaluating the toxicity and antiplasmodial activity of Momordica balsamina extracts against Plasmodium berghei. Methods: Successive extraction technique via maceration was employed. Acute oral toxicity study was conducted as per the standard protocol. In vivo antiplasmodial activity of M. balsamina extracts against early infection was assessed in P. berghei-infected mice at different doses of 100, 200, and 400 mg/kg b.w. Data was analyzed using GraphPad Prism version 8.0.2 using analysis of variance (ANOVA) followed by Tukey’s post hoc test with p

Malaria parasites, Plasmodium spp, can develop resistance to anti-malarial drugs. Hence, potential alternative therapeutic agents should be explored. This research aimed to investigate the anti-malarial effect of a combination of Carica papaya and Moringa oleifera leaf extract. Peter’s test performed on Balb/c mice to investigate the malarial parasite growth inhibition. Mice were infected with Plasmodium berghei by intraperitoneal injection to provoke rodent malaria. After infection, we grouped every 5-6 mice into different treatment groups, including a negative control group, a positive control group treated with artesunate, a moringa group, a papaya group, and a combination therapy group. Treatments were initiated 3 hours after infection (day 0) and continued every day until day 3. On day 4, we examined a thin smear of the tail vein blood for parasitemia to calculate the suppression rate. The result shows that the parasite suppression rate of C. papaya leaf extract (CPLE) is 15.02 % (percent parasitemia 4.56±1.96), the suppression rate of M. oleifera leaf extract (MOLE) is 17.32% (percent parasitemia 4.44±1.78) and that of the combination extract (CE) is 28.73% (percent parasitemia 3.82±1.48). In conclusion, there is a synergism of the anti-malarial effect of the combined leaf extract of C. papaya and M. oleifera.

Background. Phytochemicals are considered the reliable source for the treatment of infection including malaria. Especially, phenols are known as potentially toxic to the growth and development of pathogens, among which flavonoids are the most extensively studied and play more intensive roles in ethnopharmacology. The immunological effect and role of T. diversifolia flavonoids‐rich extract in treatment of malaria have therefore been examined in this study. Methods. In vitro test against Plasmodium falciparum and 4‐day suppressive and Rane's tests against Plasmodium berghei in mice were used to evaluate the antimalarial activities. TNF‐α and INF‐γ levels, phagocytic tests, and production of oxygen and nitrogen radical were assessed to appreciate the immunomodulatory activity. One‐way analysis of variance followed by post hoc Student′s t tests was used for data analysis. Results. T. diversifolia flavonoids‐rich extract at the concentrations ranging from 0.0004 mg/ml significantly (p < 0.05) inhibited in a concentration‐dependent manner the growth of trophozoite up to 100% inhibition with 0.025 mg/ml at 24 and 48 hrs. Moreover, T. diversifolia flavonoids‐rich extract reduced the level of parasitemia and improved in a dose‐dependent manner the survival time of infected mice significantly (p < 0.05) compared to their control in 4‐day suppressive test as well as in Rane's test. Additionally, T. diversifolia flavonoids‐rich extract increased the TNF‐α and INF‐γ levels in rats infected by P. berghei. Furthermore, the flavonoid‐rich extract enhanced weight of spleen in the rats, the metabolic and phagocytic activities of the peritoneal cells, and the concentration of nitric oxide and oxygen radicals in methylprednisolone‐immunocompromised rats compared to the control (p < 0.05). Conclusion. The study has revealed that T. diversifolia flavonoids‐rich extract through its antiplasmodial and phagocytic activities is a promising treatment of malaria. [ABSTRACT FROM AUTHOR]

Background Plasmodium falciparum and Plasmodium vivax account for >90% global malaria burden. Transmission intervention strategies encompassing transmission-blocking vaccines (TBV) and drugs represent ideal public health tools to eliminate malaria at the population level. The availability of mature P. falciparum gametocytes through in vitro culture has facilitated development of a standard membrane feeding assay to assess efficacy of transmission interventions against P. falciparum. The lack of in vitro culture for P. vivax has significantly hampered similar progress on P. vivax and limited studies have been possible using blood from infected patients in endemic areas. The ethical and logistical limitations of on-time access to blood from patients have impeded the development of P. vivax TBVs. Methods Transgenic murine malaria parasites (Plasmodium berghei) expressing TBV candidates offer a promising alternative for evaluation of P. vivax TBVs through in vivo studies in mice, and ex vivo membrane feeding assay (MFA). Results We describe the development of transmission-competent transgenic TgPbvs25 parasites and optimization of parameters to establish an ex vivo MFA to evaluate P. vivax TBV based on Pvs25 antigen. Conclusions The MFA is expected to expedite Pvs25-based TBV development without dependence on blood from P. vivax -infected patients in endemic areas for evaluation. [ABSTRACT FROM AUTHOR]