{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001202","ANALYSIS_ID":"AN002000","VERSION":"1","CREATED_ON":"June 25, 2019, 7:46 am"},

"PROJECT":{"PROJECT_TITLE":"System-wide biochemical analysis reveals ozonide and artemisinin antimalarials initially act by disrupting malaria parasite haemoglobin digestion","PROJECT_SUMMARY":"Artemisinins are currently the first-line antimalarials, and rely on a peroxide pharmacophore for their potent activity. OZ277 (arterolane) and OZ439 (artefenomel) are newer synthetic peroxide-based antimalarials with potent activity against the deadliest malaria parasite, Plasmodium falciparum. Here we used a “multi-omics” workflow, in combination with activity-based protein profiling (ABPP), to demonstrate that peroxide antimalarials initially target the haemoglobin (Hb) digestion pathway to kill malaria parasites. Time-dependent metabolomic profiling of peroxide-treated P. falciparum infected red blood cells (iRBCs) revealed a rapid depletion of short Hb-derived peptides, while untargeted peptidomics showed accumulation of longer Hb peptides. Quantitative proteomics and ABPP assays demonstrated that Hb digesting proteases were significantly increased in abundance and activity following treatment, respectively. The association between peroxide activity and Hb catabolism was also confirmed in a K13-mutant artemisinin resistant parasite line. To demonstrate that compromised Hb catabolism may be a primary mechanism involved in peroxide antimalarial activity, we showed that parasites forced to rely solely on Hb digestion for amino acids became hypersensitive to short peroxide exposures. Quantitative proteomics analysis also revealed parasite proteins involved in translation and the ubiquitin-proteasome system were enriched following drug treatment, suggestive of the parasite engaging a stress response to mitigate peroxide-induced damage. Taken together, these data point to a mechanism of action involving initial impairment of Hb catabolism, and indicate that the parasite regulates protein turnover to manage peroxide-induced damage.","INSTITUTE":"Monash University","LAST_NAME":"Giannangelo","FIRST_NAME":"Carlo","ADDRESS":"381 Royal Parade, Parkville, Victoria, 3052, Australia","EMAIL":"carlo.giannangelo@monash.edu","PHONE":"99039282"},

"STUDY":{"STUDY_TITLE":"Peroxide antimalarial treatment timecourse on ring-stage P. falciparum parasites","STUDY_SUMMARY":"Red blood cells (RBCs) infected with ring stage P. falciparum parasites (3D7 strain) at 10% parasitaemia and 2% haematocrit were treated with OZ277 (1 uM), OZ439 (1 uM), DHA (300 nM) or vehicle (0.03% DMSO). This was a 5-timepoint study, with samples taken 0, 1.5, 3, 6 and 9 h after drug or vehicle addition. Samples treated with vehicle acted as the untreated control. Samples from drug treated uninfected RBCs were also taken to ensure the observed drug effects were parasite specific.","INSTITUTE":"Monash University","LAST_NAME":"Giannangelo","FIRST_NAME":"Carlo","ADDRESS":"381 Royal Parade, Parkville, Victoria, 3052, Australia","EMAIL":"carlo.giannangelo@monash.edu","PHONE":"99039282"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Plasmodium falciparum;Homo sapiens","TAXONOMY_ID":"5833/9606","GENOTYPE_STRAIN":"3D7","AGE_OR_AGE_RANGE":"6-12 h post invasion"},
"SUBJECT_SAMPLE_FACTORS":[
{
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"Sample ID":"DHA_i0h_a",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i0h_a",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i0h_a",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i0h_a",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_u0h_a",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u0h_a",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u0h_a",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_i1_5h_a",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"1.5"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i1_5h_a",
"Factors":{"cell_type":"iRBC","treatment":"DMSO","treatment_duration_(h)":"1.5"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_u1_5h_a",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"1.5"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u1_5h_a",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_i3h_a",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i3h_a",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i3h_a",
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},
{
"Subject ID":"-",
"Sample ID":"DMSO_i3h_a",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_u3h_a",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u3h_a",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u3h_a",
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},
{
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},
{
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"Sample ID":"DHA_i6h_a",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i6h_a",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i6h_a",
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},
{
"Subject ID":"-",
"Sample ID":"DMSO_i6h_a",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_u6h_a",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u6h_a",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u6h_a",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u6h_a",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i9h_a",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i9h_a",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i9h_a",
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},
{
"Subject ID":"-",
"Sample ID":"OZ277_u9h_a",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u9h_a",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u9h_a",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_i0h_b",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i0h_b",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i0h_b",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i0h_b",
"Factors":{"cell_type":"iRBC","treatment":"DMSO","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_u0h_b",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u0h_b",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u0h_b",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u0h_b",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_i1_5h_b",
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},
{
"Subject ID":"-",
"Sample ID":"DMSO_i1_5h_b",
"Factors":{"cell_type":"iRBC","treatment":"DMSO","treatment_duration_(h)":"1.5"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_u1_5h_b",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"1.5"}
},
{
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"Sample ID":"DMSO_u1_5h_b",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"1.5"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_i3h_b",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i3h_b",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i3h_b",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i3h_b",
"Factors":{"cell_type":"iRBC","treatment":"DMSO","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_u3h_b",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u3h_b",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u3h_b",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u3h_b",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_i6h_b",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i6h_b",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i6h_b",
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},
{
"Subject ID":"-",
"Sample ID":"DMSO_i6h_b",
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},
{
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"Sample ID":"DHA_u6h_b",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u6h_b",
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},
{
"Subject ID":"-",
"Sample ID":"OZ439_u6h_b",
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},
{
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"Sample ID":"DMSO_u6h_b",
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},
{
"Subject ID":"-",
"Sample ID":"OZ277_i9h_b",
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},
{
"Subject ID":"-",
"Sample ID":"OZ439_i9h_b",
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},
{
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"Sample ID":"DMSO_i9h_b",
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},
{
"Subject ID":"-",
"Sample ID":"OZ277_u9h_b",
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},
{
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"Sample ID":"OZ439_u9h_b",
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},
{
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"Sample ID":"DMSO_u9h_b",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_i0h_c",
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},
{
"Subject ID":"-",
"Sample ID":"OZ277_i0h_c",
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},
{
"Subject ID":"-",
"Sample ID":"OZ439_i0h_c",
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},
{
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"Sample ID":"DMSO_i0h_c",
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},
{
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"Sample ID":"DHA_u0h_c",
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},
{
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"Sample ID":"OZ277_u0h_c",
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},
{
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"Sample ID":"OZ439_u0h_c",
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},
{
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"Sample ID":"DMSO_u0h_c",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_i1_5h_c",
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},
{
"Subject ID":"-",
"Sample ID":"DMSO_i1_5h_c",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_u1_5h_c",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"1.5"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u1_5h_c",
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},
{
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"Sample ID":"DHA_i3h_c",
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},
{
"Subject ID":"-",
"Sample ID":"OZ277_i3h_c",
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},
{
"Subject ID":"-",
"Sample ID":"OZ439_i3h_c",
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},
{
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"Sample ID":"DMSO_i3h_c",
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},
{
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"Sample ID":"DHA_u3h_c",
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},
{
"Subject ID":"-",
"Sample ID":"OZ277_u3h_c",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u3h_c",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u3h_c",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_i6h_c",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i6h_c",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i6h_c",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i6h_c",
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},
{
"Subject ID":"-",
"Sample ID":"DHA_u6h_c",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u6h_c",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u6h_c",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u6h_c",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i9h_c",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i9h_c",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i9h_c",
"Factors":{"cell_type":"iRBC","treatment":"DMSO","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u9h_c",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"9"}
},
{
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"Sample ID":"OZ439_u9h_c",
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},
{
"Subject ID":"-",
"Sample ID":"DMSO_u9h_c",
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},
{
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"Sample ID":"DHA_i0h_d",
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},
{
"Subject ID":"-",
"Sample ID":"OZ277_i0h_d",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"-"}
},
{
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"Sample ID":"OZ439_i0h_d",
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},
{
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"Sample ID":"DMSO_i0h_d",
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},
{
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"Sample ID":"DHA_u0h_d",
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},
{
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"Sample ID":"OZ277_u0h_d",
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},
{
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"Sample ID":"OZ439_u0h_d",
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},
{
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"Sample ID":"DMSO_u0h_d",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"-"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_i1_5h_d",
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},
{
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"Sample ID":"DMSO_i1_5h_d",
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},
{
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"Sample ID":"DHA_u1_5h_d",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"1.5"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u1_5h_d",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"1.5"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_i3h_d",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i3h_d",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i3h_d",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i3h_d",
"Factors":{"cell_type":"iRBC","treatment":"DMSO","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_u3h_d",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u3h_d",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u3h_d",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u3h_d",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"3"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_i6h_d",
"Factors":{"cell_type":"iRBC","treatment":"DHA","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i6h_d",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i6h_d",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i6h_d",
"Factors":{"cell_type":"iRBC","treatment":"DMSO","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"DHA_u6h_d",
"Factors":{"cell_type":"unRBC","treatment":"DHA","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u6h_d",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u6h_d",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u6h_d",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"6"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_i9h_d",
"Factors":{"cell_type":"iRBC","treatment":"OZ277","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_i9h_d",
"Factors":{"cell_type":"iRBC","treatment":"OZ439","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_i9h_d",
"Factors":{"cell_type":"iRBC","treatment":"DMSO","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"OZ277_u9h_d",
"Factors":{"cell_type":"unRBC","treatment":"OZ277","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"OZ439_u9h_d",
"Factors":{"cell_type":"unRBC","treatment":"OZ439","treatment_duration_(h)":"9"}
},
{
"Subject ID":"-",
"Sample ID":"DMSO_u9h_d",
"Factors":{"cell_type":"unRBC","treatment":"DMSO","treatment_duration_(h)":"9"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Infected RBCs were adjusted to 10% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 2E8 cells were pelleted by centrifugation at 1,000 x g for 3 min and the culture medium was removed. Parasite metabolism was quenched by the addition of ice-cold PBS, pelleted again and the supernatant discarded prior to metabolite extraction. Metabolites were extracted from the cell pellet using 200 µL of cold chloroform/methanol/water (1:3:1). The extraction solvent containing the internal standard compounds CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid) and TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) was directly added to the cell pellet, mixed by pipetting and subjected to automatic vortex mixing for 1 h at 4°C. Following the 1 h incubation, samples were pelleted by centrifugation at 21,100 x g for 10 min, 110 µL of particle free supernatant was transferred to glass LC-MS vials and stored at -80°C until analysis. A 15 µL aliquot of each sample was combined to generate a pooled biological quality control (PBQC) sample.","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Ring stage P. falciparum infected RBCs (10% parasitaemia and 2% Hct) were treated with OZ277 (1 uM), OZ439 (1 uM), DHA (300 nM) or an equivalent volume of DMSO (0.03%). DHA-treated cultures were incubated with drug for 0, 1.5, 3 and 6 h. OZ277- and OZ439-treated cultures were incubated with drug for 0, 3, 6 and 9 h. During the drug incubation period parasites were at 37°C under a gas atmosphere of 94% N2, 5% CO2 and 1% O2.","TREATMENT_COMPOUND":"OZ277 (arterolane), OZ439 (artefenomel) and dihydroartemisinin (DHA)","TREATMENT_VEHICLE":"DMSO","CELL_MEDIA":"Complete RPMI medium (10.4 g/L) containing HEPES (5.94 g/L), hypoxanthine (50 mg/L), sodium bicarbonate (2.1 g/L) and Albumax II (5 g/L).","CELL_MEDIA_LASTCHANGED":"Immediately prior to initiation of drug incubation"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Infected RBCs were adjusted to 10% parasitaemia and 2% haematocrit and the culture medium refreshed prior to drug addition. Following the drug incubation period, 2E8 cells were pelleted by centrifugation at 1,000 x g for 3 min and the culture medium was removed. Parasite metabolism was quenched by the addition of ice-cold PBS, pelleted again and the supernatant discarded prior to metabolite extraction. Metabolites were extracted from the cell pellet using 200 µL of cold chloroform/methanol/water (1:3:1). The extraction solvent containing the internal standard compounds CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid), PIPES (1,4-piperazinediethanesulfonic acid) and TRIS (2-amino-2-(hydroxymethyl)-1,3-propanediol) was directly added to the cell pellet, mixed by pipetting and subjected to automatic vortex mixing for 1 h at 4°C. Following the 1 h incubation, samples were pelleted by centrifugation at 21,100 x g for 10 min, 110 µL of particle free supernatant was transferred to glass LC-MS vials and stored at -80°C until analysis. A 15 µL aliquot of each sample was combined to generate a pooled biological quality control (PBQC) sample.","PROCESSING_STORAGE_CONDITIONS":"Described in summary"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"The 32 min gradient HPLC run was from 80% B to 50% B over 15 min, then to 5% B at 18 min, followed by a wash with 5% B for 3 min and re-equilibrated with 80% B at a flow rate of 0.3 mL/min.","CHROMATOGRAPHY_TYPE":"pHILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)","SOLVENT_A":"20 mM ammonium carbonate","SOLVENT_B":"100% acetonitrile"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"Metabolite detection was performed using a high-resolution Q Exactive MS (ThermoFisher) in both positive and negative ionisation modes. The PBQC sample was run periodically throughout each LC-MS batch to monitor signal reproducibility and support downstream metabolite identification. Extraction solvent blank samples were also analysed to identify possible contaminating chemical species. To aid in metabolite identification, approximately 250 authentic metabolite standards were analysed prior to each LC-MS batch and their peaks and retention time manually checked using the ToxID software (ThermoFisher). Metabolomics data were analysed using the IDEOM workflow (Creek et al. 2012). Briefly, the IDEOM processing pipeline uses msconvert for conversion of raw files to mzXML files and split polarity, XCMS to extract raw peak intensities and mzMatch to align samples, filter noise, fill missing peaks and annotate related peaks. Manual assessment of spiked internal standards, total ion chromatograms and median peak heights ensured signal reproducibility and allowed exclusion of outlier samples. LC MS peak heights representing metabolite abundances were normalised by median peak height. High confidence metabolite identification (MSI level 1) was made by matching accurate mass and retention time to authentic metabolite standards. Putative identifications (MSI level 2) for metabolites lacking standards were based on exact mass and predicted retention times."},

"MS_METABOLITE_DATA":{
"Units":"Peak intensity",

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}

}