In Vitro Antimalarial Activity of Trichothecenes against Liver and Blood Stages of Plasmodium Species.

Trichothecenes (TCNs) are a large group of tricyclic sesquiterpenoid mycotoxins that have intriguing structural features and remarkable biological activities. Herein, we focused on three TCNs (anguidine, verrucarin A, and verrucarol) and their ability to target both the blood and liver stages of Plasmodium species, the parasite responsible for malaria. Anguidine and verrucarin A were found to be highly effective against the blood and liver stages of malaria, while verrucarol had no effect at the highest concentration tested. However, these compounds were also found to be cytotoxic and, thus, not selective, making them unsuitable for drug development. Nonetheless, they could be useful as chemical probes for protein synthesis inhibitors due to their direct impact on parasite synthesis processes.

Cross-resistance of the chloroquine-derivative AQ-13 with amodiaquine in Cambodian Plasmodium falciparum isolates.

BACKGROUND: Expanding resistance to multiple antimalarials, including chloroquine, in South-East Asia (SEA) urges the development of new therapies. AQ-13, a chloroquine derivative, is a new drug candidate for treating malaria caused by Plasmodium falciparum. OBJECTIVES: Possible cross-resistance between the 4-aminoquinolines amodiaquine, piperaquine and AQ-13 has not been assessed. In vitro parasite growth assays were used to characterize the susceptibility of multidrug-resistant and susceptible P. falciparum patient isolates to AQ-13. METHODS: A [3H]hypoxanthine uptake assay and a 384-well high content imaging assay were used to assess efficacy of AQ-13 and desethyl-amodiaquine against 38 P. falciparum isolates. RESULTS: We observed a strong cross-resistance between the chloroquine derivative amodiaquine and AQ-13 in Cambodian P. falciparum isolates (Pearson correlation coefficient of 0.8621, P < 0.0001). CONCLUSIONS: In light of the poor efficacy of amodiaquine that we described recently in Cambodia, and its cross resistance with AQ-13, there is a significant risk that similar clinical efficacy of AQ-13-based combinations should be anticipated in areas of amodiaquine resistance.

A recombinant antibody against Plasmodium vivax UIS4 for distinguishing replicating from dormant liver stages.

BACKGROUND: Plasmodium vivax is the most geographically widespread of the human malaria parasites, causing 50,000 to 100,000 deaths annually. Plasmodium vivax parasites have the unique feature of forming dormant liver stages (hypnozoites) that can reactivate weeks or months after a parasite-infected mosquito bite, leading to new symptomatic blood stage infections. Efforts to eliminate P. vivax malaria likely will need to target the persistent hypnozoites in the liver. Therefore, research on P. vivax liver stages necessitates a marker for clearly distinguishing between actively replicating parasites and dormant hypnozoites. Hypnozoites possess a densely fluorescent prominence in the parasitophorous vacuole membrane (PVM) when stained with antibodies against the PVM-resident protein Upregulated in Infectious Sporozoites 4 (PvUIS4), resulting in a key feature recognizable for quantification of hypnozoites. Thus, PvUIS4 staining, in combination with the characteristic small size of the parasite, is currently the only hypnozoite-specific morphological marker available. RESULTS: Here, the generation and validation of a recombinant monoclonal antibody against PvUIS4 (α-rUIS4 mAb) is described. The variable heavy and light chain domains of an α-PvUIS4 hybridoma were cloned into murine IgG1 and IgK expression vectors. These expression plasmids were co-transfected into HEK293 cells and mature IgG was purified from culture supernatants. It is shown that the α-rUIS4 mAb binds to its target with high affinity. It reliably stains the schizont PVM and the hypnozoite-specific PVM prominence, enabling the visual differentiation of hypnozoites from replicating liver stages by immunofluorescence assays in different in vitro settings, as well as in liver sections from P. vivax infected liver-chimeric mice. The antibody functions reliably against all four parasite isolates tested and will be an important tool in the identification of the elusive hypnozoite. CONCLUSIONS: The α-rUIS4 mAb is a versatile tool for distinguishing replicating P. vivax liver stages from dormant hypnozoites, making it a valuable resource that can be deployed throughout laboratories worldwide.

Microphysical space of a liver sinusoid device enables simplified long-term maintenance of chimeric mouse-expanded human hepatocytes.

While many advanced liver models support hepatic phenotypes necessary for drug and disease studies, these models are characterized by intricate features such as co-culture with one of more supporting cell types or advanced media perfusion systems. These systems have helped elucidate some of the critical biophysical features missing from standard well-plate based hepatocyte culture, but their advanced designs add to their complexity. Additionally, regardless of the culture system, primary hepatocyte culture systems suffer from reproducibility issues due to phenotypic variation and expensive, limited supplies of donor lots. Here we describe a microfluidic bilayer device that sustains primary human hepatocyte phenotypes, including albumin production, factor IX production, cytochrome P450 3A4 drug metabolism and bile canaliculi formation for at least 14 days in a simple monoculture format with static media. Using a variety of channel architectures, we describe how primary cell phenotype is promoted by spatial confinement within the microfluidic channel, without the need for perfusion or co-culture. By sourcing human hepatocytes expanded in the Fah, Rag2, and Il2rg-knockout (FRG™-KO) humanized mouse model, utilizing a few hundred hepatocytes within each channel, and maintaining hepatocyte function for weeks in vitro within a relatively simple model, we demonstrate a basic primary human hepatocyte culture system that addresses many of the major hurdles in human hepatocyte culture research.

A rapid sensitive, flow cytometry-based method for the detection of Plasmodium vivax-infected blood cells.

BACKGROUND: Plasmodium vivax preferentially infects Duffy-positive reticulocytes and infections typically have few parasite-infected cells in the peripheral circulation. These features complicate detection and quantification by flow cytometry (FC) using standard nucleic acid-based staining methods. A simple antibody-based FC method was developed for rapid parasite detection along with simultaneous detection of other parasite and erythrocyte markers. METHODS: Clinical samples were collected from patients diagnosed with P. vivax at a district Malaria Clinic in Kanchanaburi, Thailand. One µL of infected blood was washed, fixed, stained with a Plasmodium pan-specific anti-PfBiP antibody conjugated with Alexa Fluor 660, and analysed by FC. Additional primary conjugated antibodies for stage-specific markers of P. vivax for late trophozoite-early schizonts (MSP1-Alexa Fluor 660), late-stage schizonts (DBP-Alexa Fluor 555), and sexual stages (Pvs16) were used to differentiate intra-erythrocytic developmental stages. RESULTS: The percentages of P. vivax-infected cells determined by the FC method and manually by microscopic examination of Giemsa-stained thick blood smears were positively correlated by Spearman's rank correlation coefficient (R2=0.93843) from 0.001 to 1.00% P. vivax-infected reticulocytes. CONCLUSIONS: The FC-based method is a simple, robust, and efficient method for detecting P. vivax-infected reticulocytes.

Sheptide A: an antimalarial cyclic pentapeptide from a fungal strain in the Herpotrichiellaceae.

As part of ongoing efforts to isolate biologically active fungal metabolites, a cyclic pentapeptide, sheptide A (1), was discovered from strain MSX53339 (Herpotrichiellaceae). The structure and sequence of 1 were determined primarily by analysis of 2D NMR and HRMS/MS data, while the absolute configuration was assigned using a modified version of Marfey's method. In an in vitro assay for antimalarial potency, 1 displayed a pEC50 value of 5.75 ± 0.49 against malaria-causing Plasmodium falciparum. Compound 1 was also tested in a counter screen for general cytotoxicity against human hepatocellular carcinoma (HepG2), yielding a pCC50 value of 5.01 ± 0.45 and indicating a selectivity factor of ~6. This makes 1 the third known cyclic pentapeptide biosynthesized by fungi with antimalarial activity.

CCR4-associated factor 1 coordinates the expression of Plasmodium falciparum egress and invasion proteins.

Coordinated regulation of gene expression is a hallmark of the Plasmodium falciparum asexual blood-stage development cycle. We report that carbon catabolite repressor protein 4 (CCR4)-associated factor 1 (CAF1) is critical in regulating more than 1,000 genes during malaria parasites' intraerythrocytic stages, especially egress and invasion proteins. CAF1 knockout results in mistimed expression, aberrant accumulation and localization of proteins involved in parasite egress, and invasion of new host cells, leading to premature release of predominantly half-finished merozoites, drastically reducing the intraerythrocytic growth rate of the parasite. This study demonstrates that CAF1 of the CCR4-Not complex is a significant gene regulatory mechanism needed for Plasmodium development within the human host.

Liver-stage fate determination in Plasmodium vivax parasites: Characterization of schizont growth and hypnozoite fating from patient isolates.

Plasmodium vivax, one species of parasite causing human malaria, forms a dormant liver stage, termed the hypnozoite, which activate weeks, months or years after the primary infection, causing relapse episodes. Relapses significantly contribute to the vivax malaria burden and are only killed with drugs of the 8-aminoquinoline class, which are contraindicated in many vulnerable populations. Development of new therapies targeting hypnozoites is hindered, in part, by the lack of robust methods to continuously culture and characterize this parasite. As a result, the determinants of relapse periodicity and the molecular processes that drive hypnozoite formation, persistence, and activation are largely unknown. While previous reports have described vastly different liver-stage growth metrics attributable to which hepatocyte donor lot is used to initiate culture, a comprehensive assessment of how different P. vivax patient isolates behave in the same lots at the same time is logistically challenging. Using our primary human hepatocyte-based P. vivax liver-stage culture platform, we aimed to simultaneously test the effects of how hepatocyte donor lot and P. vivax patient isolate influence the fate of sporozoites and growth of liver schizonts. We found that, while environmental factors such as hepatocyte donor lot can modulate hypnozoite formation rate, the P. vivax case is also an important determinant of the proportion of hypnozoites observed in culture. In addition, we found schizont growth to be mostly influenced by hepatocyte donor lot. These results suggest that, while host hepatocytes harbor characteristics making them more- or less-supportive of a quiescent versus growing intracellular parasite, sporozoite fating toward hypnozoites is isolate-specific. Future studies involving these host-parasite interactions, including characterization of individual P. vivax strains, should consider the impact of culture conditions on hypnozoite formation, in order to better understand this important part of the parasite's lifecycle.

Single-cell RNA sequencing of Plasmodium vivax sporozoites reveals stage- and species-specific transcriptomic signatures.

BACKGROUND: Plasmodium vivax sporozoites reside in the salivary glands of a mosquito before infecting a human host and causing malaria. Previous transcriptome-wide studies in populations of these parasite forms were limited in their ability to elucidate cell-to-cell variation, thereby masking cellular states potentially important in understanding malaria transmission outcomes. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we performed transcription profiling on 9,947 P. vivax sporozoites to assess the extent to which they differ at single-cell resolution. We show that sporozoites residing in the mosquito's salivary glands exist in distinct developmental states, as defined by their transcriptomic signatures. Additionally, relative to P. falciparum, P. vivax displays overlapping and unique gene usage patterns, highlighting conserved and species-specific gene programs. Notably, distinguishing P. vivax from P. falciparum were a subset of P. vivax sporozoites expressing genes associated with translational regulation and repression. Finally, our comparison of single-cell transcriptomic data from P. vivax sporozoite and erythrocytic forms reveals gene usage patterns unique to sporozoites. CONCLUSIONS/SIGNIFICANCE: In defining the transcriptomic signatures of individual P. vivax sporozoites, our work provides new insights into the factors driving their developmental trajectory and lays the groundwork for a more comprehensive P. vivax cell atlas.

Single-cell RNA profiling of Plasmodium vivax-infected hepatocytes reveals parasite- and host- specific transcriptomic signatures and therapeutic targets.

The resilience of Plasmodium vivax, the most widely-distributed malaria-causing parasite in humans, is attributed to its ability to produce dormant liver forms known as hypnozoites, which can activate weeks, months, or even years after an initial mosquito bite. The factors underlying hypnozoite formation and activation are poorly understood, as is the parasite's influence on the host hepatocyte. Here, we shed light on transcriptome-wide signatures of both the parasite and the infected host cell by sequencing over 1,000 P. vivax-infected hepatocytes at single-cell resolution. We distinguish between replicating schizonts and hypnozoites at the transcriptional level, identifying key differences in transcripts encoding for RNA-binding proteins associated with cell fate. In infected hepatocytes, we show that genes associated with energy metabolism and antioxidant stress response are upregulated, and those involved in the host immune response downregulated, suggesting both schizonts and hypnozoites alter the host intracellular environment. The transcriptional markers in schizonts, hypnozoites, and infected hepatocytes revealed here pinpoint potential factors underlying dormancy and can inform therapeutic targets against P. vivax liver-stage infection.

Cryopreservation of Plasmodium Sporozoites.

Malaria is a deadly disease caused by the parasite, Plasmodium, and impacts the lives of millions of people around the world. Following inoculation into mammalian hosts by infected mosquitoes, the sporozoite stage of Plasmodium undergoes obligate development in the liver before infecting erythrocytes and causing clinical malaria. The most promising vaccine candidates for malaria rely on the use of attenuated live sporozoites to induce protective immune responses. The scope of widespread testing or clinical use of such vaccines is limited by the absence of efficient, reliable, or transparent strategies for the long-term preservation of live sporozoites. Here we outline a method to cryopreserve the sporozoites of various human and murine Plasmodium species. We found that the structural integrity, viability, and in vivo or in vitro infectiousness were conserved in the recovered cryopreserved sporozoites. Cryopreservation using our approach also retained the transgenic properties of sporozoites and immunization with cryopreserved radiation attenuated sporozoites (RAS) elicited strong immune responses. Our work offers a reliable protocol for the long-term storage and recovery of human and murine Plasmodium sporozoites and lays the groundwork for the widespread use of live sporozoites for research and clinical applications.

Alkyne modified purines for assessment of activation of Plasmodium vivax hypnozoites and growth of pre-erythrocytic and erythrocytic stages in Plasmodium spp.

Malaria is a major global health problem which predominantly afflicts developing countries. Although many antimalarial therapies are currently available, the protozoan parasite causing this disease, Plasmodium spp., continues to evade eradication efforts. One biological phenomenon hampering eradication efforts is the parasite's ability to arrest development, transform into a drug-insensitive form, and then resume growth post-therapy. Currently, the mechanisms by which the parasite enters arrested development, or dormancy, and later recrudesces or reactivates to continue development, are unknown and the malaria field lacks techniques to study these elusive mechanisms. Since Plasmodium spp. salvage purines for DNA synthesis, we hypothesised that alkyne-containing purine nucleosides could be used to develop a DNA synthesis marker which could be used to investigate mechanisms behind dormancy. Using copper-catalysed click chemistry methods, we observe incorporation of alkyne modified adenosine, inosine, and hypoxanthine in actively replicating asexual blood stages of Plasmodium falciparum and incorporation of modified adenosine in actively replicating liver stage schizonts of Plasmodium vivax. Notably, these modified purines were not incorporated in dormant liver stage hypnozoites, suggesting this marker could be used as a tool to differentiate replicating and non-replicating liver forms and, more broadly, as a tool for advancing our understanding of Plasmodium dormancy mechanisms.

Metabolic, Pharmacokinetic, and Activity Profile of the Liver Stage Antimalarial (RC-12).

The catechol derivative RC-12 (WR 27653) (1) is one of the few non-8-aminoquinolines with good activity against hypnozoites in the gold-standard Plasmodium cynomolgi-rhesus monkey (Macaca mulatta) model, but in a small clinical trial, it had no efficacy against Plasmodium vivax hypnozoites. In an attempt to better understand the pharmacokinetic and pharmacodynamic profile of 1 and to identify potential active metabolites, we now describe the phase I metabolism, rat pharmacokinetics, and in vitro liver-stage activity of 1 and its metabolites. Compound 1 had a distinct metabolic profile in human vs monkey liver microsomes, and the data suggested that the O-desmethyl, combined O-desmethyl/N-desethyl, and N,N-didesethyl metabolites (or a combination thereof) could potentially account for the superior liver stage antimalarial efficacy of 1 in rhesus monkeys vs that seen in humans. Indeed, the rate of metabolism was considerably lower in human liver microsomes in comparison to rhesus monkey microsomes, as was the formation of the combined O-desmethyl/N-desethyl metabolite, which was the only metabolite tested that had any activity against liver-stage P. vivax; however, it was not consistently active against liver-stage P. cynomolgi. As 1 and all but one of its identified Phase I metabolites had no in vitro activity against P. vivax or P. cynomolgi liver-stage malaria parasites, we suggest that there may be additional unidentified active metabolites of 1 or that the exposure of 1 achieved in the reported unsuccessful clinical trial of this drug candidate was insufficient to kill the P. vivax hypnozoites.

Regulatory hotspots in the malaria parasite genome dictate transcriptional variation.

The determinants of transcriptional regulation in malaria parasites remain elusive. The presence of a well-characterized gene expression cascade shared by different Plasmodium falciparum strains could imply that transcriptional regulation and its natural variation do not contribute significantly to the evolution of parasite drug resistance. To clarify the role of transcriptional variation as a source of stain-specific diversity in the most deadly malaria species and to find genetic loci that dictate variations in gene expression, we examined genome-wide expression level polymorphisms (ELPs) in a genetic cross between phenotypically distinct parasite clones. Significant variation in gene expression is observed through direct co-hybridizations of RNA from different P. falciparum clones. Nearly 18% of genes were regulated by a significant expression quantitative trait locus. The genetic determinants of most of these ELPs resided in hotspots that are physically distant from their targets. The most prominent regulatory locus, influencing 269 transcripts, coincided with a Chromosome 5 amplification event carrying the drug resistance gene, pfmdr1, and 13 other genes. Drug selection pressure in the Dd2 parental clone lineage led not only to a copy number change in the pfmdr1 gene but also to an increased copy number of putative neighboring regulatory factors that, in turn, broadly influence the transcriptional network. Previously unrecognized transcriptional variation, controlled by polymorphic regulatory genes and possibly master regulators within large copy number variants, contributes to sweeping phenotypic evolution in drug-resistant malaria parasites.

Synthesis of Mono- and Bisperoxide-Bridged Artemisinin Dimers to Elucidate the Contribution of Dimerization to Antimalarial Activity.

During the past decade, artemisinin as an antimalarial has been in the spotlight, in part due to the Nobel Prize in Physiology or Medicine awarded to Tu Youyou. While many studies have been completed detailing the significant increase in activity resulting from the dimerization of natural product artemisinin, activity increases unaccounted for by the peroxide bridge have yet to be researched. Here we outline the synthesis and testing for antimalarial activity of artemisinin dimers in which the peroxide bridge in one-half of the dimer is reduced, resulting in a dimer with one active and one deactivated artemisinin moiety.

Characterization of the Tubovesicular Network in Plasmodium vivax Liver Stage Hypnozoites and Schizonts.

Plasmodium is a genus of apicomplexan parasites which replicate in the liver before causing malaria. Plasmodium vivax can also persist in the liver as dormant hypnozoites and cause clinical relapse upon activation, but the molecular mechanisms leading to activation have yet to be discovered. In this study, we use high-resolution microscopy to characterize temporal changes of the P. vivax liver stage tubovesicular network (TVN), a parasitophorous vacuole membrane (PVM)-derived network within the host cytosol. We observe extended membrane clusters, tubules, and TVN-derived vesicles present throughout P. vivax liver stage development. Additionally, we demonstrate an unexpected presence of the TVN in hypnozoites and observe some association of this network to host nuclei. We also reveal that the host water and solute channel aquaporin-3 (AQP3) associates with TVN-derived vesicles and extended membrane clusters. AQP3 has been previously shown to localize to the PVM of P. vivax hypnozoites and liver schizonts but has not yet been shown in association to the TVN. Our results highlight host-parasite interactions occur in both dormant and replicating liver stage P. vivax forms and implicate AQP3 function during this time. Together, these findings enhance our understanding of P. vivax liver stage biology through characterization of the TVN with an emphasis on the presence of this network in dormant hypnozoites.

A Phenotypic Screen for the Liver Stages of Plasmodium vivax.

Control of malaria caused by Plasmodium vivax can be improved by the discovery and development of novel drugs against the parasite's liver stage, which includes relapse-causing hypnozoites. Several recent reports describe breakthroughs in the culture of the P. vivax liver stage in 384-well microtiter plates, with the goal of enabling a hypnozoite-focused drug screen. Herein we describe assay details, protocol developments, and different assay formats to interrogate the chemical sensitivity of the P. vivax liver stage in one such medium-throughput platform. The general assay protocol includes seeding of primary human hepatocytes which are infected with P. vivax sporozoites generated from the feeding of Anopheles dirus mosquitoes on patient isolate bloodmeals. This protocol is unique in that, after source drug plates are supplied, all culture-work steps have been optimized to preclude the need for automated liquid handling, thereby allowing the assay to be performed within resource-limited laboratories in malaria-endemic countries. Throughput is enhanced as complex culture methods, such as extracellular matrix overlays, multiple cell types in co-culture, or hepatic spheroids, are excluded as the workflow consists entirely of routine culture methods for adherent cells. Furthermore, installation of a high-content imager at the study site enables assay data to be read and transmitted with minimal logistical delays. Herein we detail distinct assay improvements which increase data quality, provide a means to limit the confounding effect of hepatic metabolism on assay data, and detect activity of compounds with a slow-clearance phenotype. Graphical abstract: Overview of P. vivax liver stage screening assay performed at the Institute Pasteur of Cambodia.

Aminoalkoxycarbonyloxymethyl Ether Prodrugs with a pH-Triggered Release Mechanism: A Case Study Improving the Solubility, Bioavailability, and Efficacy of Antimalarial 4(1H)-Quinolones with Single Dose Cures.

Preclinical and clinical development of numerous small molecules is prevented by their poor aqueous solubility, limited absorption, and oral bioavailability. Herein, we disclose a general prodrug approach that converts promising lead compounds into aminoalkoxycarbonyloxymethyl (amino AOCOM) ether-substituted analogues that display significantly improved aqueous solubility and enhanced oral bioavailability, restoring key requirements typical for drug candidate profiles. The prodrug is completely independent of biotransformations and animal-independent because it becomes an active compound via a pH-triggered intramolecular cyclization-elimination reaction. As a proof-of-concept, the utility of this novel amino AOCOM ether prodrug approach was demonstrated on an antimalarial compound series representing a variety of antimalarial 4(1H)-quinolones, which entered and failed preclinical development over the last decade. With the amino AOCOM ether prodrug moiety, the 3-aryl-4(1H)-quinolone preclinical candidate was shown to provide single-dose cures in a rodent malaria model at an oral dose of 3 mg/kg, without the use of an advanced formulation technique.

Probing the distinct chemosensitivity of Plasmodium vivax liver stage parasites and demonstration of 8-aminoquinoline radical cure activity in vitro.

Improved control of Plasmodium vivax malaria can be achieved with the discovery of new antimalarials with radical cure efficacy, including prevention of relapse caused by hypnozoites residing in the liver of patients. We screened several compound libraries against P. vivax liver stages, including 1565 compounds against mature hypnozoites, resulting in one drug-like and several probe-like hits useful for investigating hypnozoite biology. Primaquine and tafenoquine, administered in combination with chloroquine, are currently the only FDA-approved antimalarials for radical cure, yet their activity against mature P. vivax hypnozoites has not yet been demonstrated in vitro. By developing an extended assay, we show both drugs are individually hypnozonticidal and made more potent when partnered with chloroquine, similar to clinically relevant combinations. Post-hoc analyses of screening data revealed excellent performance of ionophore controls and the high quality of single point assays, demonstrating a platform able to support screening of greater compound numbers. A comparison of P. vivax liver stage activity data with that of the P. cynomolgi blood, P. falciparum blood, and P. berghei liver stages reveals overlap in schizonticidal but not hypnozonticidal activity, indicating that the delivery of new radical curative agents killing P. vivax hypnozoites requires an independent and focused drug development test cascade.

Plasmodium vivax Liver and Blood Stages Recruit the Druggable Host Membrane Channel Aquaporin-3.

Plasmodium vivax infects hepatocytes to form schizonts that cause blood infection, or dormant hypnozoites that can persist for months in the liver before leading to relapsing blood infections. The molecular processes that drive P. vivax schizont and hypnozoite survival remain largely unknown, but they likely involve a rich network of host-pathogen interactions, including those occurring at the host-parasite interface, the parasitophorous vacuole membrane (PVM). Using a recently developed P. vivax liver-stage model system we demonstrate that host aquaporin-3 (AQP3) localizes to the PVM of schizonts and hypnozoites within 5 days after invasion. This recruitment is also observed in P. vivax-infected reticulocytes. Chemical treatment with the AQP3 inhibitor auphen reduces P. vivax liver hypnozoite and schizont burden, and inhibits P. vivax asexual blood-stage growth. These findings reveal a role for AQP3 in P. vivax liver and blood stages and suggest that the protein may be targeted for therapeutic treatment.