{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001057","ANALYSIS_ID":"AN001726","VERSION":"1","CREATED_ON":"September 19, 2018, 10:18 am"},

"PROJECT":{"PROJECT_TITLE":"Improved Extraction method of Polar and Charged Metabolites from Cyanobacteria.","PROJECT_SUMMARY":"We report excellent extraction method for cyanobacteria of the genus Synechococcus, viz., Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 7942, and a newly isolated Synechococcus elongatus PCC 11801 (manuscript under review). High resolution-LC/MS was used to assess the relative abundance of the extracted metabolites. The different solvent systems used for extraction led to statistically significant changes in the extraction efficiency for a large number of metabolites. While a few hundred m/z features or potential metabolites were detected with different solvent systems, the abundance of over a quarter of all metabolites varied significantly from one solvent system to another. Further, the extraction methods were evaluated for a targeted set of metabolites that are important in 13C-MFA studies of photosynthetic organisms. While for the strain PCC 7002, the reported method using methanol-chloroform-water system gave satisfactory results, a mild base in the form of NH4OH had to be used in place of water to achieve adequate levels of extraction for PCC 7942 and PCC 11801.","INSTITUTE":"Indian Institute of Technology, Bombay","DEPARTMENT":"Department of Chemical Engineering","LABORATORY":"Wangikar Laboratory","LAST_NAME":"Wangikar","FIRST_NAME":"Pramod","ADDRESS":"Room No. 136, Biosystems Lab","EMAIL":"wangikar@iitb.ac.in","PHONE":"02225764215"},

"STUDY":{"STUDY_TITLE":"Metabolite Extractions from Cyanobacteria","STUDY_SUMMARY":"Metabolite Extractions data from the Strains Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 11801 (New strain, Manuscript submitted) and Synechococcus elongatus PCC 7942","INSTITUTE":"Indian Institute of Technology, Bombay","DEPARTMENT":"Department of Chemical Engineering","LABORATORY":"Wangikar Laboratory","LAST_NAME":"Wangikar","FIRST_NAME":"Pramod","ADDRESS":"Room No. 136, Biosystems Lab","EMAIL":"wangikar@iitb.ac.in","PHONE":"2225764215"},

"SUBJECT":{"SUBJECT_TYPE":"Bacteria","SUBJECT_SPECIES":"Synechococcus","TAXONOMY_ID":"1117"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 1_Rep1",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"1"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 1_Rep2",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"2"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 1_Rep3",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"3"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 1_Rep4",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"4"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 2A_Rep1",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"5"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 2A_Rep2",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"6"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 2A_Rep3",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"7"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 2B_0.4M NH4OH_Rep1",
"Factors":{"Extraction Method Used":"Method 2B-0.4M NH4OH"},
"Additional sample data":{"No.":"8"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 2B_0.4M NH4OH_Rep2",
"Factors":{"Extraction Method Used":"Method 2B-0.4M NH4OH"},
"Additional sample data":{"No.":"9"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 2B_0.4M NH4OH_Rep3",
"Factors":{"Extraction Method Used":"Method 2B-0.4M NH4OH"},
"Additional sample data":{"No.":"10"}
},
{
"Subject ID":"Synechococcus sp. PCC 7002",
"Sample ID":"PCC 7002_Method 2B_0.4M NH4OH_Rep4",
"Factors":{"Extraction Method Used":"Method 2B-0.4M NH4OH"},
"Additional sample data":{"No.":"11"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 1_Rep1",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"12"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 1_Rep2",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"13"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 1_Rep3",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"14"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 2A_Rep1",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"15"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 2A_Rep2",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"16"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 2A_Rep3",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"17"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 2A_Rep4",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"18"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 2B_0.4M NH4OH_Rep1",
"Factors":{"Extraction Method Used":"Method 2B-0.4M NH4OH"},
"Additional sample data":{"No.":"19"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 2B_0.4M NH4OH_Rep2",
"Factors":{"Extraction Method Used":"Method 2B-0.4M NH4OH"},
"Additional sample data":{"No.":"20"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 2B_0.4M NH4OH_Rep3",
"Factors":{"Extraction Method Used":"Method 2B-0.4M NH4OH"},
"Additional sample data":{"No.":"21"}
},
{
"Subject ID":"Synechococcus elongatus PCC 7942",
"Sample ID":"PCC 7942_Method 2B_0.4M NH4OH_Rep4",
"Factors":{"Extraction Method Used":"Method 2B-0.4M NH4OH"},
"Additional sample data":{"No.":"22"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 1_Rep1",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"23"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 1_Rep2",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"24"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 1_Rep3",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"25"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 1_Rep4",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"26"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 1_Rep5",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"27"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 1_Rep6",
"Factors":{"Extraction Method Used":"Method 1"},
"Additional sample data":{"No.":"28"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2A_Rep1",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"29"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2A_Rep2",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"30"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2A_Rep3",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"31"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2A_Rep4",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"32"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2A_Rep5",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"33"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2A_Rep6",
"Factors":{"Extraction Method Used":"Method 2A"},
"Additional sample data":{"No.":"34"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2B-0.2M NH4OH_Rep1",
"Factors":{"Extraction Method Used":"Method 2B-0.2M NH4OH"},
"Additional sample data":{"No.":"35"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2B-0.2M NH4OH_Rep2",
"Factors":{"Extraction Method Used":"Method 2B-0.2M NH4OH"},
"Additional sample data":{"No.":"36"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2B-0.2M NH4OH_Rep3",
"Factors":{"Extraction Method Used":"Method 2B-0.2M NH4OH"},
"Additional sample data":{"No.":"37"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2B-0.2M NH4OH_Rep4",
"Factors":{"Extraction Method Used":"Method 2B-0.2M NH4OH"},
"Additional sample data":{"No.":"38"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2B-0.2M NH4OH_Rep5",
"Factors":{"Extraction Method Used":"Method 2B-0.2M NH4OH"},
"Additional sample data":{"No.":"39"}
},
{
"Subject ID":"Synechococcus elongatus PCC 11801",
"Sample ID":"PCC 11801_Method 2B-0.2M NH4OH_Rep6",
"Factors":{"Extraction Method Used":"Method 2B-0.2M NH4OH"},
"Additional sample data":{"No.":"40"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"20 mL Cyanobacterial cells grown at exponential phase were filtered and extracted with the extraction buffers suggested.","SAMPLE_TYPE":"Bacterial cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"Various cells were extracted using the extraction solutions described as Method 1, Method 2A and Method 2B"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The cell suspension was filtered through a 0.8 µm nylon membrane and the filtered cells was quickly placed in 1.6 ml cold methanol (100%) or methanol-water mixture (80:20 v/v). The mixture was incubated at -80 °C for 1 hour following which the cell-solvent mixture was removed and placed in a 15 mL centrifuge tube. The cells sticking to the filter were then scraped off with chloroform (2.4 mL) and mixed with the cell-solvent mixture. The mixture was vortexed for 20 minutes under cold conditions (vortexed for 30s and kept on ice for 1 minute and the cycle repeated). Two mL of MilliQ water or NH4OH solution in water (at concentrations of 0.2 M, 0.4 M) was added into the mixture to separate the phases. The mixture was vortexed for 10 minutes, centrifuged for 15 minutes at 3200g at 4 °C to obtain two separate phases and the top aqueous-rich layer was collected (approximately 3 mL), evaporated to dryness and stored at -80 °C until further analysis. The stored pellets were reconstituted in 100 μL of 50/50 methanol-water mixture and filtered before injecting on LC/MS."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Shimadzu 20AD","COLUMN_NAME":"Phenomenex Synergi Hydro RP (100 x 2mm, 2.5um)","FLOW_RATE":"300 microliter/min","SOLVENT_A":"10 mMTributylamine","SOLVENT_B":"Methanol"},

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

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"ABI Sciex 5600+ TripleTOF","INSTRUMENT_TYPE":"Triple TOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE"},

"MS_METABOLITE_DATA":{
"Units":"Peak Area",

"Data":[{"Metabolite":"Lactate","PCC 7002_Method 1_Rep1":"","PCC 7002_Method 1_Rep2":"","PCC 7002_Method 1_Rep3":"","PCC 7002_Method 1_Rep4":"","PCC 7002_Method 2A_Rep1":"","PCC 7002_Method 2A_Rep2":"","PCC 7002_Method 2A_Rep3":"","PCC 7002_Method 2B_0.4M NH4OH_Rep1":"","PCC 7002_Method 2B_0.4M NH4OH_Rep2":"","PCC 7002_Method 2B_0.4M NH4OH_Rep3":"","PCC 7002_Method 2B_0.4M NH4OH_Rep4":"","PCC 7942_Method 1_Rep1":"1852.9359","PCC 7942_Method 1_Rep2":"1402.4217","PCC 7942_Method 1_Rep3":"1648.5385","PCC 7942_Method 2A_Rep1":"","PCC 7942_Method 2A_Rep2":"","PCC 7942_Method 2A_Rep3":"","PCC 7942_Method 2A_Rep4":"","PCC 7942_Method 2B_0.4M NH4OH_Rep1":"","PCC 7942_Method 2B_0.4M NH4OH_Rep2":"","PCC 7942_Method 2B_0.4M NH4OH_Rep3":"","PCC 7942_Method 2B_0.4M NH4OH_Rep4":"","PCC 11801_Method 1_Rep1":"899.8988","PCC 11801_Method 1_Rep2":"1375.1727","PCC 11801_Method 1_Rep3":"792.0333","PCC 11801_Method 1_Rep4":"792.8255","PCC 11801_Method 1_Rep5":"1258.476","PCC 11801_Method 1_Rep6":"1301.5295","PCC 11801_Method 2A_Rep1":"892.8924","PCC 11801_Method 2A_Rep2":"858.8369","PCC 11801_Method 2A_Rep3":"865.0828","PCC 11801_Method 2A_Rep4":"893.6553","PCC 11801_Method 2A_Rep5":"919.8789","PCC 11801_Method 2A_Rep6":"1005.3058","PCC 11801_Method 2B-0.2M NH4OH_Rep1":"1791.6433","PCC 11801_Method 2B-0.2M NH4OH_Rep2":"1888.1907","PCC 11801_Method 2B-0.2M NH4OH_Rep3":"1675.5431","PCC 11801_Method 2B-0.2M NH4OH_Rep4":"1713.6814","PCC 11801_Method 2B-0.2M NH4OH_Rep5":"1563.2564","PCC 11801_Method 2B-0.2M NH4OH_Rep6":"1592.7803"},{"Metabolite":"Proglutamic Acid","PCC 7002_Method 1_Rep1":"","PCC 7002_Method 1_Rep2":"","PCC 7002_Method 1_Rep3":"","PCC 7002_Method 1_Rep4":"","PCC 7002_Method 2A_Rep1":"","PCC 7002_Method 2A_Rep2":"","PCC 7002_Method 2A_Rep3":"","PCC 7002_Method 2B_0.4M NH4OH_Rep1":"","PCC 7002_Method 2B_0.4M NH4OH_Rep2":"","PCC 7002_Method 2B_0.4M NH4OH_Rep3":"","PCC 7002_Method 2B_0.4M NH4OH_Rep4":"","PCC 7942_Method 1_Rep1":"","PCC 7942_Method 1_Rep2":"","PCC 7942_Method 1_Rep3":"","PCC 7942_Method 2A_Rep1":"","PCC 7942_Method 2A_Rep2":"","PCC 7942_Method 2A_Rep3":"","PCC 7942_Method 2A_Rep4":"","PCC 7942_Method 2B_0.4M NH4OH_Rep1":"","PCC 7942_Method 2B_0.4M NH4OH_Rep2":"","PCC 7942_Method 2B_0.4M NH4OH_Rep3":"","PCC 7942_Method 2B_0.4M NH4OH_Rep4":"","PCC 11801_Method 1_Rep1":"534.5225","PCC 11801_Method 1_Rep2":"1093.8752","PCC 11801_Method 1_Rep3":"373.2025","PCC 11801_Method 1_Rep4":"348.0604","PCC 11801_Method 1_Rep5":"971.2025","PCC 11801_Method 1_Rep6":"1008.7229","PCC 11801_Method 2A_Rep1":"117.5174","PCC 11801_Method 2A_Rep2":"1295.8125","PCC 11801_Method 2A_Rep3":"1291.3516","PCC 11801_Method 2A_Rep4":"438.8461","PCC 11801_Method 2A_Rep5":"653.476","PCC 11801_Method 2A_Rep6":"675.0187","PCC 11801_Method 2B-0.2M NH4OH_Rep1":"713.38","PCC 11801_Method 2B-0.2M NH4OH_Rep2":"1179.1403","PCC 11801_Method 2B-0.2M NH4OH_Rep3":"843.8781","PCC 11801_Method 2B-0.2M NH4OH_Rep4":"681.9004","PCC 11801_Method 2B-0.2M NH4OH_Rep5":"813.329","PCC 11801_Method 2B-0.2M NH4OH_Rep6":"715.1182"},{"Metabolite":"Malate","PCC 7002_Method 1_Rep1":"13810.0856","PCC 7002_Method 1_Rep2":"13270.3789","PCC 7002_Method 1_Rep3":"13308.8749","PCC 7002_Method 1_Rep4":"14280.9022","PCC 7002_Method 2A_Rep1":"15786.2019","PCC 7002_Method 2A_Rep2":"13366.3427","PCC 7002_Method 2A_Rep3":"13057.5638","PCC 7002_Method 2B_0.4M NH4OH_Rep1":"12590.7215","PCC 7002_Method 2B_0.4M NH4OH_Rep2":"11738.5977","PCC 7002_Method 2B_0.4M NH4OH_Rep3":"13260.6195","PCC 7002_Method 2B_0.4M NH4OH_Rep4":"12619.877","PCC 7942_Method 1_Rep1":"","PCC 7942_Method 1_Rep2":"","PCC 7942_Method 1_Rep3":"","PCC 7942_Method 2A_Rep1":"","PCC 7942_Method 2A_Rep2":"","PCC 7942_Method 2A_Rep3":"","PCC 7942_Method 2A_Rep4":"","PCC 7942_Method 2B_0.4M NH4OH_Rep1":"","PCC 7942_Method 2B_0.4M NH4OH_Rep2":"","PCC 7942_Method 2B_0.4M NH4OH_Rep3":"","PCC 7942_Method 2B_0.4M NH4OH_Rep4":"","PCC 11801_Method 1_Rep1":"90.3848","PCC 11801_Method 1_Rep2":"202.1407","PCC 11801_Method 1_Rep3":"323.1465","PCC 11801_Method 1_Rep4":"337.4176","PCC 11801_Method 1_Rep5":"223.5786","PCC 11801_Method 1_Rep6":"232.9166","PCC 11801_Method 2A_Rep1":"300.3222","PCC 11801_Method 2A_Rep2":"1338.547","PCC 11801_Method 2A_Rep3":"1399.5292","PCC 11801_Method 2A_Rep4":"1789.7308","PCC 11801_Method 2A_Rep5":"2175.8477","PCC 11801_Method 2A_Rep6":"2381.9141","PCC 11801_Method 2B-0.2M NH4OH_Rep1":"1412.2336","PCC 11801_Method 2B-0.2M NH4OH_Rep2":"1445.9477","PCC 11801_Method 2B-0.2M NH4OH_Rep3":"1331.5346","PCC 11801_Method 2B-0.2M NH4OH_Rep4":"1353.2254","PCC 11801_Method 2B-0.2M NH4OH_Rep5":"1372.5855","PCC 11801_Method 2B-0.2M NH4OH_Rep6":"1341.7374"},{"Metabolite":"Glutamate","PCC 7002_Method 1_Rep1":"1841.0787","PCC 7002_Method 1_Rep2":"1768.5991","PCC 7002_Method 1_Rep3":"1899.9246","PCC 7002_Method 1_Rep4":"1785.2176","PCC 7002_Method 2A_Rep1":"2615.2457","PCC 7002_Method 2A_Rep2":"1984.8242","PCC 7002_Method 2A_Rep3":"1526.1567","PCC 7002_Method 2B_0.4M NH4OH_Rep1":"564.2993","PCC 7002_Method 2B_0.4M NH4OH_Rep2":"491.6767","PCC 7002_Method 2B_0.4M NH4OH_Rep3":"490.4675","PCC 7002_Method 2B_0.4M NH4OH_Rep4":"856.9978","PCC 7942_Method 1_Rep1":"2283.5649","PCC 7942_Method 1_Rep2":"2602.931","PCC 7942_Method 1_Rep3":"2583.6524","PCC 7942_Method 2A_Rep1":"1989.7402","PCC 7942_Method 2A_Rep2":"1220.2165","PCC 7942_Method 2A_Rep3":"1326.8634","PCC 7942_Method 2A_Rep4":"1015.7544","PCC 7942_Method 2B_0.4M NH4OH_Rep1":"992.2812","PCC 7942_Method 2B_0.4M NH4OH_Rep2":"1370.9715","PCC 7942_Method 2B_0.4M NH4OH_Rep3":"1144.985","PCC 7942_Method 2B_0.4M NH4OH_Rep4":"1312.5851","PCC 11801_Method 1_Rep1":"7772.6607","PCC 11801_Method 1_Rep2":"8886.6645","PCC 11801_Method 1_Rep3":"8650.4606","PCC 11801_Method 1_Rep4":"8393.6557","PCC 11801_Method 1_Rep5":"7905.4443","PCC 11801_Method 1_Rep6":"8569.6907","PCC 11801_Method 2A_Rep1":"7352.6305","PCC 11801_Method 2A_Rep2":"6716.5347","PCC 11801_Method 2A_Rep3":"5891.3158","PCC 11801_Method 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}

}