For: Pesticide biochemistry and physiology recG is involved with the resistance of Bt to UV.

As an ATP-dependent DNA helicase, RecG can repair DNA replication forks in many organisms. However, knowledge of recG in Bacillus thuringiensis (Bt) is limited. In our previous study, recG was found damaged in Bt LLP29-M19, which was more resistant to ultraviolet light (UV) after exposing Bt LLP29 to UV for 19 generations. To further understand the function of recG in the mechanism of Bt UV resistance, recG was knocked out and recovered with homologous recombination technology in Bt LLP29. Comparing the resistance of the different mutants to UVB, Bt ∆recG-LLP29 lacking recG was found more sensitive to UVB, hydroxyurea (HU) and H2O2 than LLP29 and the complementation strain. To compare the expression level of recG in the Bt strains under different UV treatments, Quantitative Real-time PCR (RT-qPCR) of recG was performed in the tested Bt strains, which showed that the expression level of recG in Bt ∆recG-LLP29 was substantially lower than that in the original strain and complementation strain. Interestingly, when exposed to UV for 20 min, RecG expression in both Bt LLP29 and Bt recG-R was the highest. The unwinding activity of recG in Bt LLP29 and the complementation strain were also found higher than that of the recG knockout strain, Bt ∆recG-LLP29. These results demonstrate that recG is involved with the resistance of Bt to UV. These findings not only enhance the understanding of the Bt UV resistance mechanism, but also provide an important theoretical basis for the application of Bt.

Heterologous expression and purification of BtCspB, a novel cold-shock protein-like bacteriocin from Bacillus thuringiensis BRC-ZYR2.

A novel Bacillus thuringiensis (Bt) bacteriocin BtCspB, active against a food-borne pathogen Bacillus cereus, was identified and purified by a traditional four-step chromatographic process with low yield (44.5 µg/L) in our lab previously. The aim of this study was to dramatically increase its yield by heterologous expression of BtCspB. The BtCspB gene from Bt BRC-ZYR2 was successfully heterologously expressed in Escherichia coli BL21 (DE3). Affinity chromatography was used to obtain the pure BtCspB up to 20 mg/L. The purified BtCspB showed a MIC value of 12.5 µg/mL and a MBC value of 50.0 µg/mL against Bacillus cereus ATCC 10987. The bacteriocin activity of BtCspB against B. cereus ATCC 10987 was further directly detected in a gel-overlay assay. The anti-B. cereus activity, however, was lower than the bacteriocin purified by the traditional four-step chromatographic process probably because of structural modifications. Compared with the traditional method, the yield of the bacteriocin by heterologous expression increased by 449 times, and the purification step was dramatically simplified, which laying a foundation for the industrial production of this novel cold-shock protein-like bacteriocin BtCspB active against B. cereus.

Nematicidal Activity of Cry1Ea11 from Bacillus thuringiensis BRC-XQ12 Against the Pine Wood Nematode (Bursaphelenchus xylophilus).

The nematicidal activity of 92 Bacillus thuringiensis strains against the pine wood nematode Bursaphelenchus xylophilus, one of the world's top 10 plant-parasitic nematodes, was determined. The insecticidal crystal proteins (ICPs) from Bacillus thuringiensis BRC-XQ12 were the most toxic to Bursaphelenchus xylophilus, with a lethal concentration 50 (LC50) of 32.13 µg/ml. Because the ICPs expressed by Bacillus thuringiensis BRC-XQ12 were closest to Cry1Ea6 and B. thuringiensis BRC-XQ12 contained four kinds of cry1 subgenes (cry1Aa, cry1Cb, cry1Ea, and cry1Ia), Cry1Ea was most likely to be the key active component against the nematode. The 3,516-bp cry1Ea11 gene from BRC-XQ12, as designated by the B. thuringiensis δ-endotoxin nomenclature committee, was expressed in Escherichia coli. Purified Cry1Ea11 showed an LC50 of 32.53 and 23.23 µg/ml at 24 and 48 h, with corresponding virulence equations of Y = 32.15X + 1.38 (R2 = 0.9951) and Y = 34.29X + 3.16 (R2 = 0.9792), respectively. In order to detect the pathway of B. thuringiensis Cry1Ea11 into Bursaphelenchus xylophilus, the nematode was fed with NHS-rhodamine-labeled GST-Cry1Ea11. The results of confocal laser-scanning microscopy showed that the 159-kDa GST-Cry1Ea11 could be detected in the stylet and the esophageal lumen of the pine wood nematode, indicating that GST-Cry1Ea11 could enter into the nematode through the stylet. As far as we know, no Cry1 proteins have been shown to have activity against plant-parasitic nematodes before. These results demonstrate that Cry1Ea11 is a promising nematicidal protein for controlling pine wilt disease rendered by B. xylophilus, further dramatically broadening the spectrum of Bacillus thuringiensis ICPs.

In vitro and in vivo efficacies of novel carbazole aminoalcohols in the treatment of cystic echinococcosis.

OBJECTIVES: Cystic echinococcosis (CE), caused by the cestode Echinococcus granulosus, is a worldwide chronic zoonosis. Current chemotherapeutic options are limited to albendazole and mebendazole, which only exert parasitostatic effects and have to be administered at high dosages for long periods. In an effort to find alternative treatment options, the in vitro and in vivo efficacies of novel carbazole aminoalcohols were evaluated. METHODS: Carbazole aminoalcohols were tested against E. granulosus protoscoleces in vitro and metacestodes ex vivo. The in vivo chemotherapeutic effect of representative compounds was assessed in experimentally infected mice. Oral and intravenous pharmacokinetic profiles were determined in mice. RESULTS: The carbazole aminoalcohols exhibited potent protoscolicidal activity with LC50 values ranging from 18.2 to 34.3 µM. Among them, compounds 2 and 24 killed all ex vivo cultured metacestodes at concentrations of 34.3 and 30.6 µM. In vivo studies showed that oral administration of compounds 2 and 24 (25 mg/kg/day) for 30 days led to reductions of 68.4% and 54.3% in parasite weight compared with the untreated group (both groups: P < 0.001). Compound 2 (25 mg/kg/day) and compound 24 (50 mg/kg/day) induced significantly higher cyst mortality rates in comparison with that of the albendazole group (both groups: P < 0.01). Analysis of cysts collected from compound 2- or 24-treated mice by transmission electron microscopy revealed a drug-induced structural destruction. The structural integrity of the germinal layer was lost, and the majority of the microtriches disappeared. Pharmacokinetic profiling of compounds 2 and 24 revealed low clearance and decent oral bioavailability (>70%). CONCLUSIONS: Our study identifies carbazole aminoalcohols as a class of novel anti-CE agents. Compounds 2 and 24 represent promising drug candidates in anti-CE chemotherapy.

c-di-AMP accumulation impairs toxin expression of Bacillus anthracis by down-regulating potassium importers.

The Gram-positive bacterium Bacillus anthracis is the causative agent of anthrax and a bioterrorism threat worldwide. As a crucial second messenger in many bacterial species, cyclic di-AMP (c-di-AMP) modulates various key processes for bacterial homeostasis and pathogenesis. Overaccumulation of c-di-AMP alters cellular growth and reduces anthrax toxin expression as well as virulence in Bacillus anthracis by unresolved underlying mechanisms. In this report, we discovered that c-di-AMP binds to a series of receptors involved in potassium uptake in B. anthracis. By analyzing Kdp and Ktr mutants for osmotic stress, gene expression, and anthrax toxin expression, we also showed that c-di-AMP inhibits Kdp operon expression through binding to the KdpD and ydaO riboswitch; up-regulating intracellular potassium promotes anthrax toxin expression in c-di-AMP accumulated B. anthracis. Decreased anthrax toxin expression at high c-di-AMP occurs through the inhibition of potassium uptake. Understanding the molecular basis of how potassium uptake affects anthrax toxin has the potential to provide new insight into the control of B. anthracis.IMPORTANCEThe bacterial second messenger cyclic di-AMP (c-di-AMP) is a conserved global regulator of potassium homeostasis. How c-di-AMP regulates bacterial virulence is unknown. With this study, we provide a link between potassium uptake and anthrax toxin expression in Bacillus anthracis. c-di-AMP accumulation might inhibit anthrax toxin expression by suppressing potassium uptake.

Enhanced bioavailability and cysticidal effect of three mebendazole-oil preparations in mice infected with secondary cysts of Echinococcus granulosus.

The aim of the present study is to explore the possibility to increase the efficacy of mebendazole (MBZ) against secondary cysts of Echinococcus granulosus harbored in mice by augmenting the solubility and bioavailability of the drug. Firstly, the saturated solubility of MBZ in nine kinds of oil was determined by high performance liquid chromatography (HPLC), and MBZ was found exhibiting the highest, secondary, and lowest solubility in oleic acid (OA), glycerol trioleate (GT), and soybean oil (SB), respectively. Secondly, MBZ-OA suspension, MBZ-GT suspension, MBZ-SB suspension, and MBZ suspended in 1 % tragacanth (MBZ-1 % tragacanth) were selected for further studies on pharmacokinetics and experimental therapy in mice. Four groups of mice were treated orally with one of aforementioned four MBZ preparations at a single dose of 25 mg/kg, and concentrations of MBZ in plasma obtained from each mouse at various intervals within 24 h postadministration were determined by HPLC. The major pharmacokinetic parameters calculated by MBZ plasma concentration-time curve demonstrated that the peak concentration of the drug (C (max) ) values obtained from three MBZ-oil preparation groups was 1.6-2.8 times higher than that of MBZ-1 % tragacanth group. The same was true that the area under the drug concentration-time curve (AUC(0-∞)) values of 19.8 (2.5)-28.2 (2.5) µg/ml × h revealed in the three MBZ-oil preparation groups was significantly higher than that of 11.6 (2.0) µg/ml × h in MBZ-1 % tragacanth group, and the bioavailability of the three MBZ-oil preparation groups was 71-143 % higher than that of MBZ-1 % tragacanth group. In mice infected with secondary cysts of E. granulosus for 8 months treated orally with MBZ-1 % tragacanth at a daily dose of 25 mg/kg for 14 consecutive days, the mean cyst weight was lower than that of untreated control, but the difference was not statistically significant with cyst weight reduction of 48 %. When the infected mice received three MBZ-oil preparations at the same oral dose schedule as aforementioned, the mean cyst weights were significantly lower than those in MBZ-1 % tragacanth group or control group with cyst weight reductions of 71.2-84.7 %. The results indicate that the solubility of MBZ in oils may increase to various degrees according to the kinds of oil used. Meanwhile, three MBZ-oil (OA, GT, and SB) preparations administered orally to mice not only improve the bioavailability of MBZ relative to that of MBZ suspended in 1 % tragacanth, but their effects against hydatid cysts also significantly enhance.

Comparative proteomics suggests the mode of action of a novel molluscicide against the invasive apple snail Pomacea canaliculata, intermediate host of Angiostrongylus cantonensis.

Angiostrongylus cantonensis is a zoonotic parasitic nematode that is the most common cause of human eosinophilic meningitis. The invasive apple snail Pomacea canaliculata is an important intermediate host of A. cantonensis and contributes to its spread. P. canaliculata control will help prevent its invasion and transmission of A. cantonensis. The new molluscicide PBQ (1-(4-chlorophenyl)-3-(pyridin-3-yl)urea) exhibits great potency against P. canaliculata and has low toxicity against mammals and non-target aquatic organisms. We studied the mode of action of PBQ using TMT-based comparative quantitative proteomics analysis between PBQ-treated and control P. canaliculata snails. A total of 3151 proteins were identified, and 245 of these proteins were significantly differentially expressed with 135 downregulated and 110 upregulated. GO and KEGG enrichment analyses identified GO terms and KEGG pathways involved in de novo purine biosynthesis, ribosome components and translation process were significantly enriched and downregulated. The results indicated that PBQ treatment had substantial effects on the synthesis of genetic material, translation process, and protein synthesis of P. canaliculata and were likely the main cause of snail mortality.

Optimization of an Evaluation Method for Anti-Babesia microti Drug Efficacy.

Babesiosis is an emerging zoonotic disease that is typically caused by Babesia microti infection. Clinical treatment of B. microti infection is challenging; hence, it is crucial to find new effective drugs. The current laboratory screening methods for anti-B. microti drugs are not optimized. We conducted drug-suppressive and drug-therapeutic tests to investigate whether use of an immunosuppressant and the target gene-based qPCR are helpful to reduce the number of animals affected and to improve parasite detection in an immunocompetent mouse model. These results were verified by subpassage test. In the drug-suppressive test, no B. microti were observed after immunosuppressant administration or in subpassage mice in the 100 mg/kg robenidine hydrochloride (ROBH) group. The opposite results were observed in the control, 50 mg/kg ROBH, atovaquone (ATO) + azithromycin (AZM), and proguanil hydrochloride (PGH) groups. Significant differences were observed in the EIR and target gene relative values (both P < 0.001) between the control group and any ROBH groups. In the drug-therapeutic test, recrudescence occurred in the 50 mg/kg ROBH, ATO+AZM, and control groups. This was not observed in the 100 mg/kg ROBH group after immunosuppressant administration. Similar findings were observed in the subpassage test. This suggests that a 4-day anti-B. microti drug-suppressive test can be used in preliminary drug screening. Potentially effective drugs can be verified by immunosuppressant test in subsequent drug-therapeutic tests. Thus, a laboratory evaluation method of anti-B. microti drug efficacy was optimized, which is highly accurate and requires a short drug screening time.

Control of the Invasive Agricultural Pest Pomacea canaliculata with a Novel Molluscicide: Efficacy and Safety to Nontarget Species.

The golden apple snail Pomacea canaliculata is an invasive pest that causes extensive damage to agricultural production. P. canaliculata is also an intermediate host of Angiostrongylus cantonensis, which causes human eosinophilic meningitis. In this study, the molluscicidal activity and safety profile of a novel molluscicide PBQ [1-(4-chlorophenyl)-3-(pyridin-3-yl)urea] were evaluated. PBQ exhibited strong molluscicidal potency against adult and juvenile snails (LC50 values of 0.39 and 0.07 mg/L, respectively). In field trials, PBQ killed 99.42% of the snails at 0.25 g a.i./m2. An acute toxicity test in rats demonstrated that PBQ is a generally nonhazardous chemical. PBQ is also generally safe for nontarget organisms including Brachydanio rerio, Daphnia magna, and Apis mellifera L. Transcriptomics analysis revealed that PBQ had a significant impact on the carbohydrate and lipid metabolism pathways, which provided insights into its molluscicidal mechanism. These results suggest that PBQ could be developed as an effective and safe molluscicide for P. canaliculata control.

Identification of an orally active carbazole aminoalcohol derivative with broad-spectrum anti-animal trypanosomiasis activity.

Animal trypanosomiasis, caused by the members of subgenus Trypanozoon (Trypanosoma brucei brucei, T. evansi and T. equiperdum), has reduced animal productivity leading to significant negative economic impacts in endemic regions. Due to limited drug discovery and the emergence of drug-resistance over many recent decades, novel and effective compounds against animal trypanosomiasis are urgently required. This study was conducted to evaluate the antitrypanosomal potential of a batch of carbazole aminoalcohol derivatives. Among them, we found that the most effective compound was H1402, which exhibited potent trypanocidal efficacy against the bloodstream-form of T. b. brucei (EC50 = 0.73 ± 0.05 µM) and presented low cytotoxicity against two mammalian cell lines with CC50 > 30 µM. Using a murine model of acute infection, oral administration with H1402 demonstrated a complete clearance of T. b. brucei and all the infected mice were cured when they were treated twice daily for 5 days at a dose of 100 mg/kg. Furthermore, parasites were not detected in mice infected with T. evansi and T. equiperdum (the causative agents of surra and dourine, respectively, in animals) within 30 days following the same regimen with H1402. In addition, H1402 caused severe morphological and ultrastructural destruction to trypanosomes, as well as causing phosphatidylserine externalization, which are suggested to be the most likely cause of cell death. Overall, the present data demonstrated that H1402 could be promising as a rapid, safe and orally active lead compound for the development of new chemotherapeutics for animal trypanosomiasis.

Methnaridine is an orally bioavailable, fast-killing and long-acting antimalarial agent that cures Plasmodium infections in mice.

BACKGROUND AND PURPOSE: Malaria is one of the deadliest diseases in the world. Novel chemotherapeutic agents are urgently required to combat the widespread Plasmodium resistance to frontline drugs. Here, we report the discovery of a novel benzonaphthyridine antimalarial, methnaridine, which was identified using a structural optimization strategy. EXPERIMENTAL APPROACH: An integrated pharmacological approach was used to evaluate the antimalarial profile of methnaridine. The pharmacokinetic properties of methnaridine were investigated along with the associated safety profile. Host immune response patterns were also analysed. KEY RESULTS: Methnaridine exhibited potent antimalarial activity against P. falciparum (3D7: IC50 = 0.0066 µM; Dd2: IC50 = 0.0056 µM). In P. berghei-infected mice, oral administration effectively suppressed parasitemia (ED50 = 0.52 mg·kg-1 ·day-1 ) and cured the established infection (CD50 = 10.13 mg·kg-1 ·day-1 ). These results are equivalent to or better than those of other antimalarial agents in clinical use. Notably, a four-dose oral regimen at a dosage of 25 mg·kg-1 achieved a complete cure of P. berghei infection in mice. Methnaridine exhibited a rapid parasiticidal profile (PCT99 = 36.0 h) and showed no cross-resistance to chloroquine. Pharmacokinetic studies revealed that methnaridine is readily absorbed, long-lasting and slowly cleared. The safety profile of methnaridine is also satisfactory (maximum tolerated dose = 1,125 mg·kg-1 ). In addition, following methnaridine treatment, infection-induced Th1 immune response was almost fully alleviated in mice. CONCLUSION AND IMPLICATIONS: Methnaridine is an orally bioavailable, fast-acting and long-lasting agent with excellent antimalarial properties. Our study highlights the potential of methnaridine for clinical development as a promising antimalarial candidate.

Old drug repurposing for neglected disease: Pyronaridine as a promising candidate for the treatment of Echinococcus granulosus infections.

BACKGROUND: Cystic echinococcosis (CE), a condition caused by the larval stage of the dog tapeworm Echinococcus granulosus sensu stricto, is a globally distributed zoonotic disease. Current treatment options for CE are limited, and an effective and safe anti-echinococcal drug is urgently required. METHODS: Drug repurposing strategy was employed to identify new therapeutic agents against echinococcal cysts. An in vitro protoscolicidal assay along with in vivo murine models was applied in the drug screening. A microinjection procedure was employed to mimic the clinical PAIR (puncture, aspiration, injection and reaspiration) technique to evaluate the potential application of the candidate drug in clinical practice. FINDINGS: We repurposed pyronaridine, an approved antimalarial drug, for the treatment of CE. Following a three-dose intraperitoneal regimen (57 mg/kg, q.d. for 3 days), pyronaridine caused 100% cyst mortality. Oral administration of pyronaridine at 57 mg/kg, q.d. for 30 days significantly reduced the parasitic burden in the pre-infected mice compared with albendazole group (p < 0.001). Using a microinjection of drug into cysts, pyronaridine (200 µM) showed highly effective in term of inhibition of cyst growth (p < 0.05, compared with saline group). Pharmacokinetic analysis revealed that pyronaridine was highly distributed in the liver and lungs, the most affected organs of CE. Function analysis showed that pyronaridine inhibited the activity of topoisomerase I (IC50 = 209.7 ± 1.1 µM). In addition, classical apoptotic hallmarks, including DNA fragmentation and caspase activation, were triggered. INTERPRETATION: Given its approved clinical safety, the repurposing of pyronaridine offers a rapidly translational option for treating CE including PAIR. FUND: National Natural Science Foundation of China and International Cooperation Project of the Qinghai Science and Technology Department.

Toxicity of a molluscicide candidate PPU07 against Oncomelania hupensis (Gredler, 1881) and local fish in field evaluation.

Schistosomiasis japonica caused by Schistosoma japonicum infection is recognized as a considerable economic and public health concern in Asia. Oncomelania hupensis is the sole intermediate host of S. japonicum. The only molluscicide recommended by World Health Organization (WHO) since 1960s is relative toxic to other aquatic species. In this article, we evaluated the novel molluscicide PPU07 in field trials on their efficiency against O. hupensis and toxicity for local fish. 25% PPU07 sulfate WP exhibited similar molluscicidal effect at 2.0 g/m2 and 2.0 g/m3 in the spraying and immersion trials with the WHO recommended molluscicide niclosamide (1 g/m2 and 1 g/m3). The mortality rates reached 95% and 96%, respectively. Moreover, little toxicity was observed for local fish and other aquatic organisms at the effective molluscicidal concentrations. In all, 25% PPU07 sulfate WP is a promising molluscicide for snail control, particularly in semi-commercial or commercial aquaculture ponds.

Discovery of the pyridylphenylureas as novel molluscicides against the invasive snail Biomphalaria straminea, intermediate host of Schistosoma mansoni.

BACKGROUND: The snail Biomphalaria straminea is one of the intermediate hosts of Schistosoma mansoni. Biomphalaria straminea is also an invasive species, known for its strong capability on peripheral expansion, long-distance dispersal and colonization. Using molluscicides to control snail populations is an important strategy to interrupt schistosomiasis transmission and to prevent the spread of the invasive species. In this study, a series of pyridylphenylurea derivatives were synthesized as potential molluscicides. Their impact on adult snails and egg masses was evaluated. Acute toxicity to fish of the derivatives was also examined to assess their effect on non-target organisms. The preliminary mechanisms of action of the derivatives were studied by enzyme activity assays. RESULTS: The representative compounds, 1-(4-chlorophenyl)-3-(pyridin-3-yl)urea (compound 8) and 1-(4-bromophenyl)-3-(pyridin-3-yl)urea (compound 9), exhibited strong molluscicidal activity against adult snails with LD50 values of 0.50 and 0.51 mg/l and potent inhibitory effects on snail egg hatchability with IC50 values of 0.05 and 0.09 mg/l. Notably, both compounds showed good target specificity with potent molluscicidal capability observed in snails, but very low toxicity to local fishes. Furthermore, the exposure of compounds 8 and 9 significantly elevated the enzyme activities of acid phosphatase and nitric oxide synthase of the snails, while no significant change was recorded in the activities of alkaline phosphatase, acetylcholine esterase and superoxide dismutase. CONCLUSION: The results suggested that compounds 8 and 9 of pyridylphenylurea derivatives could be developed as promising molluscicide candidates for snail control.

Carbazole Aminoalcohols Induce Antiproliferation and Apoptosis of Human Tumor Cells by Inhibiting Topoisomerase†I.

Novel carbazole aminoalcohols were designed and synthesized as anticancer agents. Among them, alkylamine-chain-substituted compounds showed the most promising antiproliferative activity, with IC50 values in the single-digit micromolar range against two human tumor cell lines. Topoisomerase I (topo I) is likely to be one of the targets of these compounds. Results of comet assays and molecular docking indicate that the representative compounds may act as topo I poisons, causing single-strand DNA damage by stabilizing the topo I-DNA cleavage complex. In particular, the most potent compound, 1-(butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (6), was shown to be able to induce G2 -phase cell-cycle arrest and apoptosis in HeLa cells.

[Field evaluation of IgG antibody detection kits of Schistosoma japonicum and quality control analysis].

OBJECTIVE: To evaluate the application effect of an ELISA kit for detection of IgG antibody to Schistosomajaponicum in endemic areas. METHODS: In two schistosomiasis endemic villages of Eryuan County, Yunnan Province, there were 505 permanent residents enrolled in the study. Fecal examinations were carried out by using the nylon screen bag-egg concentration method. In paralleled testing, the ELISA kit was used for the villagers to detect the IgG antibody to S. japonicum. The assay was performed daily for 20 consecutive days, and the data were analyzed by comparison with the parasitological examination and by using the L - J quality control chart method. RESULTS: In 505 examinees, 290 were positive by ELISA, with a positive rate of 57.43%. The L-J chart analysis showed that the deviation of A450 value between the quality control sera A and B was within the controllable range. The fecal examination found 20 positives with a positive rate 3.96%. Compared with the fecal examination, the positive consistency rate of the ELISA method reached to 90.00%. CONCLUSION: The ELISA kit for detection of IgG antibody to S. japonicum is stable, reliable and suitable for the screening of schistosomiasis in the field.

Selection and reversal of Plasmodium berghei resistance in the mouse model following repeated high doses of artemether.

Artemether, a derivative of artemisinin, is effectively used for the treatment of malaria without any clinically relevant resistance to date. Artemether has also been developed as an antischistosomal agent, exhibiting highest activity against immature parasites. Here, we employ a rodent model and investigate whether the proposed artemether treatment schedule to prevent schistosome-attributable morbidity might select for Plasmodium berghei resistance. Mice infected with an ANKA strain of P. berghei were treated with artemether at either 47 mg/kg or 300 mg/kg. Once every 7-10 days, parasitized erythrocytes were passed to the next group of mice, receiving the same doses of artemether, for 50 passages. Resistance development was slow but increased considerably over the final ten passages. At the higher dose of artemether, the indices of resistance were 4.8 and 8.8 after 40 and 50 passages, respectively. Importantly, resistance was unstable, since sensitivity reverted to near-normal after five passages without drug pressure. A moderate index of P. berghei resistance and no apparent reversibility was found in comparative experiments employing pyronaridine. In conclusion, the pace of resistance development in P. berghei to repeated high doses of artemether is slow and reversible.