#METABOLOMICS WORKBENCH eallman_20200525_112201 DATATRACK_ID:2020 STUDY_ID:ST001384 ANALYSIS_ID:AN002308 PROJECT_ID:PR000948 VERSION 1 CREATED_ON May 26, 2020, 10:06 am #PROJECT PR:PROJECT_TITLE Plasmodium falciparum increased time in circulation underlies persistent PR:PROJECT_TITLE asymptomatic infection in the dry season PR:PROJECT_SUMMARY The dry season is a major challenge for Plasmodium falciparum parasites in many PR:PROJECT_SUMMARY malaria endemic regions, where water availability limits mosquitoes to only part PR:PROJECT_SUMMARY of the year. How P. falciparum bridges two transmission seasons months apart, PR:PROJECT_SUMMARY without being cleared by the host or compromising host survival is poorly PR:PROJECT_SUMMARY understood. Here we show that low levels of P. falciparum parasites persist in PR:PROJECT_SUMMARY the blood of asymptomatic Malian individuals during the 5- to 6-month dry PR:PROJECT_SUMMARY season, rarely causing symptoms and minimally affecting the host immune PR:PROJECT_SUMMARY response. Parasites isolated during the dry season are transcriptionally PR:PROJECT_SUMMARY distinct from those of subjects with febrile malaria in the transmission season, PR:PROJECT_SUMMARY reflecting longer circulation within each replicative cycle, of parasitized PR:PROJECT_SUMMARY erythrocytes without adhering to the vascular endothelium. Low parasite levels PR:PROJECT_SUMMARY during the dry season are not due to impaired replication, but rather increased PR:PROJECT_SUMMARY splenic clearance of longer-circulating infected erythrocytes. We propose that PR:PROJECT_SUMMARY P. falciparum virulence in areas of seasonal malaria transmission is regulated PR:PROJECT_SUMMARY so that the parasite decreases its endothelial binding capacity, allowing PR:PROJECT_SUMMARY increased splenic clearance and enabling several months of subclinical parasite PR:PROJECT_SUMMARY persistence. PR:INSTITUTE Penn State PR:LAST_NAME Llinas PR:FIRST_NAME Manuel PR:ADDRESS W126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA PR:EMAIL manuel@psu.edu PR:PHONE (814) 867-3527 #STUDY ST:STUDY_TITLE Plasmodium falciparum increased time in circulation underlies persistent ST:STUDY_TITLE asymptomatic infection in the dry season ST:STUDY_SUMMARY The dry season is a major challenge for Plasmodium falciparum parasites in many ST:STUDY_SUMMARY malaria endemic regions, where water availability limits mosquitoes to only part ST:STUDY_SUMMARY of the year. How P. falciparum bridges two transmission seasons months apart, ST:STUDY_SUMMARY without being cleared by the host or compromising host survival is poorly ST:STUDY_SUMMARY understood. Here we show that low levels of P. falciparum parasites persist in ST:STUDY_SUMMARY the blood of asymptomatic Malian individuals during the 5- to 6-month dry ST:STUDY_SUMMARY season, rarely causing symptoms and minimally affecting the host immune ST:STUDY_SUMMARY response. Parasites isolated during the dry season are transcriptionally ST:STUDY_SUMMARY distinct from those of subjects with febrile malaria in the transmission season, ST:STUDY_SUMMARY reflecting longer circulation within each replicative cycle, of parasitized ST:STUDY_SUMMARY erythrocytes without adhering to the vascular endothelium. Low parasite levels ST:STUDY_SUMMARY during the dry season are not due to impaired replication, but rather increased ST:STUDY_SUMMARY splenic clearance of longer-circulating infected erythrocytes. We propose that ST:STUDY_SUMMARY P. falciparum virulence in areas of seasonal malaria transmission is regulated ST:STUDY_SUMMARY so that the parasite decreases its endothelial binding capacity, allowing ST:STUDY_SUMMARY increased splenic clearance and enabling several months of subclinical parasite ST:STUDY_SUMMARY persistence. ST:INSTITUTE Penn State ST:LAST_NAME Llinas ST:FIRST_NAME Manuel ST:ADDRESS W126 Millennium Science Complex, University Park, PENNSYLVANIA, 16802, USA ST:EMAIL manuel@psu.edu ST:PHONE (814) 867-3527 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #FACTORS #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - a244 Genotype:May RAW_FILE_NAME=a244 SUBJECT_SAMPLE_FACTORS - a308 Genotype:May RAW_FILE_NAME=a308 SUBJECT_SAMPLE_FACTORS - a320 Genotype:May RAW_FILE_NAME=a320 SUBJECT_SAMPLE_FACTORS - a325 Genotype:May RAW_FILE_NAME=a325 SUBJECT_SAMPLE_FACTORS - a326 Genotype:May RAW_FILE_NAME=a326 SUBJECT_SAMPLE_FACTORS - a334 Genotype:May RAW_FILE_NAME=a334 SUBJECT_SAMPLE_FACTORS - a351 Genotype:May RAW_FILE_NAME=a351 SUBJECT_SAMPLE_FACTORS - a355 Genotype:May RAW_FILE_NAME=a355 SUBJECT_SAMPLE_FACTORS - a357 Genotype:May RAW_FILE_NAME=a357 SUBJECT_SAMPLE_FACTORS - a373 Genotype:May RAW_FILE_NAME=a373 SUBJECT_SAMPLE_FACTORS - a388 Genotype:May RAW_FILE_NAME=a388 SUBJECT_SAMPLE_FACTORS - a448 Genotype:May RAW_FILE_NAME=a448 SUBJECT_SAMPLE_FACTORS - b161 Genotype:MAL RAW_FILE_NAME=b161 SUBJECT_SAMPLE_FACTORS - b170 Genotype:MAL RAW_FILE_NAME=b170 SUBJECT_SAMPLE_FACTORS - b224 Genotype:MAL RAW_FILE_NAME=b224 SUBJECT_SAMPLE_FACTORS - b262 Genotype:MAL RAW_FILE_NAME=b262 SUBJECT_SAMPLE_FACTORS - b322 Genotype:MAL RAW_FILE_NAME=b322 SUBJECT_SAMPLE_FACTORS - b332 Genotype:MAL RAW_FILE_NAME=b332 SUBJECT_SAMPLE_FACTORS - b371 Genotype:MAL RAW_FILE_NAME=b371 SUBJECT_SAMPLE_FACTORS - b385 Genotype:MAL RAW_FILE_NAME=b385 SUBJECT_SAMPLE_FACTORS - b391 Genotype:MAL RAW_FILE_NAME=b391 SUBJECT_SAMPLE_FACTORS - b400 Genotype:MAL RAW_FILE_NAME=b400 SUBJECT_SAMPLE_FACTORS - b443 Genotype:MAL RAW_FILE_NAME=b443 SUBJECT_SAMPLE_FACTORS - b459 Genotype:MAL RAW_FILE_NAME=b459 SUBJECT_SAMPLE_FACTORS - poola1 Genotype:May Pool RAW_FILE_NAME=poola1 SUBJECT_SAMPLE_FACTORS - poola2 Genotype:May Pool RAW_FILE_NAME=poola2 SUBJECT_SAMPLE_FACTORS - poola3 Genotype:May Pool RAW_FILE_NAME=poola3 SUBJECT_SAMPLE_FACTORS - poolb1 Genotype:MAL Pool RAW_FILE_NAME=poolb1 SUBJECT_SAMPLE_FACTORS - poolb2 Genotype:MAL Pool RAW_FILE_NAME=poolb2 SUBJECT_SAMPLE_FACTORS - poolb3 Genotype:MAL Pool RAW_FILE_NAME=poolb3 SUBJECT_SAMPLE_FACTORS - qc1 Genotype:Pool RAW_FILE_NAME=qc1 SUBJECT_SAMPLE_FACTORS - qc2 Genotype:Pool RAW_FILE_NAME=qc2 SUBJECT_SAMPLE_FACTORS - qc3 Genotype:Pool RAW_FILE_NAME=qc3 SUBJECT_SAMPLE_FACTORS - blank1 Genotype:Blank RAW_FILE_NAME=blank1 SUBJECT_SAMPLE_FACTORS - blank2 Genotype:Blank RAW_FILE_NAME=blank2 SUBJECT_SAMPLE_FACTORS - blank3 Genotype:Blank RAW_FILE_NAME=blank3 SUBJECT_SAMPLE_FACTORS - blank4 Genotype:Blank RAW_FILE_NAME=blank4 #COLLECTION CO:COLLECTION_SUMMARY 2 mL venous blood was drawn of RDT+ individuals tested at the end of the dry CO:COLLECTION_SUMMARY season (May 2012) cross-sectional, and at the first malaria episode of the CO:COLLECTION_SUMMARY ensuing transmission season, into EDTA tubes (Vacutainer K3EDTA Tubes, BD) and CO:COLLECTION_SUMMARY processed directly at the field site. Plasma (used for metabolomic analysis) was CO:COLLECTION_SUMMARY separated by centrifugation and immediately frozen in liquid N2. Buffy coat was CO:COLLECTION_SUMMARY discarded and the RBC pellet was further removed of leucocytes in a two-step CO:COLLECTION_SUMMARY procedure; first by density gradient on Lymphoprep solution (Fresenius Kabi), CO:COLLECTION_SUMMARY followed by Plasmodipur (EuroProxima) filtration. CO:SAMPLE_TYPE Blood (plasma) #TREATMENT TR:TREATMENT_SUMMARY None, samples were collected at 2 natural timepoints. RDT+ individuals tested at TR:TREATMENT_SUMMARY the end of the dry season (May 2012) cross-sectional, and at the first malaria TR:TREATMENT_SUMMARY episode of the ensuing transmission season. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Each plasma sample was split into two independent samples for metabolite SP:SAMPLEPREP_SUMMARY extraction. For hydrophilic metabolites, 50µL of plasma was extracted by the SP:SAMPLEPREP_SUMMARY addition of 9X volumes of ice cold methanol. Samples were briefly vortexed SP:SAMPLEPREP_SUMMARY before centrifuging for 10 minutes to remove precipitated protein. The clarified SP:SAMPLEPREP_SUMMARY supernatants were dried under nitrogen gas and resuspended in 100µL (1:2 SP:SAMPLEPREP_SUMMARY dilution final). For hydrophobic metabolites, 25µL of plasma was extracted by SP:SAMPLEPREP_SUMMARY the addition of 3X volumes of isopropanol. Samples were briefly vortexed and SP:SAMPLEPREP_SUMMARY allowed to sit at room temperature for 10 minutes. Samples were then placed at SP:SAMPLEPREP_SUMMARY -20 °C to precipitate overnight. Precipitated samples were centrifuged for 20 SP:SAMPLEPREP_SUMMARY minutes and the clarified supernatant was diluted to 50% water in a glass LCMS SP:SAMPLEPREP_SUMMARY sample vial (1:6 dilution final). Sample groups were pooled to create a group QA SP:SAMPLEPREP_SUMMARY and all samples were pooled to create a batch QC, which were injected SP:SAMPLEPREP_SUMMARY periodically throughout each run. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Shimadzu Prominence 20 UFLCXR CH:COLUMN_NAME Waters BEH C18 (100mm x 2.1mm; 1.7 µm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME ABI Sciex 5600 TripleTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The capillary voltage was set at 4.5 kV in negative ion mode, with a MS:MS_COMMENTS declustering potential of 80V. The mass spectrometer was operated in IDA MS:MS_COMMENTS (Information Dependent Acquisition) mode with a 100 ms survey scan from 100 to MS:MS_COMMENTS 1200 m/z, and up to 20 MS/MS product ion scans (100 ms) per duty cycle using a MS:MS_COMMENTS collision energy of 50V with a 20V spread. Data processing was performed using MS:MS_COMMENTS MS-DIAL and annotated using the built-in public library. Blank subtraction and MS:MS_COMMENTS analysis was performed in excel. MS:MS_RESULTS_FILE ST001384_AN002308_Results.txt UNITS:Area Has m/z:Yes Has RT:Yes RT units:Minutes #END