{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002877","ANALYSIS_ID":"AN004714","VERSION":"1","CREATED_ON":"10-04-2023"},

"PROJECT":{"PROJECT_TITLE":"Exogenous L-Alanine promotes phagocytosis via dual regulations of TLR4 to eliminate multidrug-resistant bacterial pathogens","PROJECT_TYPE":"MS quantitative analysis","PROJECT_SUMMARY":"Multidrug-resistant bacteria present a major threat to public health. Therefore, new drugs or approaches are urgently needed to manage and mitigate this threat. Here, we screen the molecular candidates that allow the survival of mice upon multidrug-resistant Vibrio parahaemolyticus infection by integrated proteomic and metabolomics analysis, where L-Alanine metabolism and phagocytosis are highly correlated. The role of L-Alanine on boosting mouse survival is further confirmed with in vivo bacterial challenge studies on multidrug-resistant bacteria including V. parahaemolyticus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae. Functional studies demonstrate that exogenous L-Alanine promotes phagocytosis to these different species of multidrug-resistant pathogens. The underlying mechanism involves two events that are L-Alanine-dependently increased TLR4 expression, and L-Alanine-enhanced TLR4 signaling via increasing the biosynthesis and secretion of fatty acids such as palmitate. Palmitate enhances the binding of LPS to TLR4 and thereby promotes TLR4 dimmer formation and endocytosis for the subsequent activation of PI3K/Akt and NF-κB pathways and phagocytosis of bacteria. These results suggest that modulation of metabolic environment is a plausible approach for combating infection with multidrug-resistant bacteria.","INSTITUTE":"sun yat-sen university","LAST_NAME":"jiang","FIRST_NAME":"ming","ADDRESS":"No. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China, guangzhou, guangdong, 510006, China","EMAIL":"jiangm28@mail.sysu.edu.cn","PHONE":"13434283781","DOI":"http://dx.doi.org/10.21228/M85M79"},

"STUDY":{"STUDY_TITLE":"Metabolic Profiling of Raw264.7 Mouse Macrophage Cells Cultured with Alanine","STUDY_SUMMARY":"To identify the catabolites of L-Alanine on promoting phagocytosis, GC-MS based metabolomics analysis was adopted to explore L-Alanine-reprogrammed metabolome. The metabolic flow of the TCA cycle was dysregulated. Meanwhile, six metabolites (oleate, palmitate, stearate, myristate, arachidonate and linoleate) in biosynthesis of saturated and unsaturated fatty acids were increased upon L-Alanine treatment, where palmitate was the biggest absolute increment in abundance. Thus, L-Alanine promotes the biosynthesis of fatty acids.","INSTITUTE":"Sun Yat-sen University","LAST_NAME":"jiang","FIRST_NAME":"ming","ADDRESS":"No. 135, Xingang Xi Road, Guangzhou, 510275, P. R. China, guangzhou, guangdong, 510006, China","EMAIL":"jiangm28@mail.sysu.edu.cn","PHONE":"13434283781","SUBMIT_DATE":"2023-09-14"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Ala-40-1-1",
"Factors":{"factor":"Ala"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"Ala-40-1-1.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"Ala-40-1-2",
"Factors":{"factor":"Ala"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"Ala-40-1-2.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"Ala-40-2-1",
"Factors":{"factor":"Ala"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"Ala-40-2-1.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"Ala-40-2-2",
"Factors":{"factor":"Ala"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"Ala-40-2-2.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"Ala-40-3-1",
"Factors":{"factor":"Ala"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"Ala-40-3-1.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"Ala-40-3-2",
"Factors":{"factor":"Ala"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"Ala-40-3-2.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"control-1-1",
"Factors":{"factor":"Control"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"control-1-1.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"control-1-2",
"Factors":{"factor":"Control"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"control-1-2.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"control-2-1",
"Factors":{"factor":"Control"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"control-2-1.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"control-2-2",
"Factors":{"factor":"Control"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"control-2-2.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"control-3-1",
"Factors":{"factor":"Control"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"control-3-1.raw","RAW_FILE_NAME":"-"}
},
{
"Subject ID":"-",
"Sample ID":"control-3-2",
"Factors":{"factor":"Control"},
"Additional sample data":{"other":"Wild-type","RAW_FILE_NAME":"control-3-2.raw","RAW_FILE_NAME":"-"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Cells were counted, washed with cold PBS and then flash-frozen in liquid N2","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"To trace L-Alanine metabolism, RAW264.7 cell were grown in DMEM (Hyclone) supplemented with 10% (v/v) cosmic calf (Hyclone), then transferred into L-Alanine-free medium and deprived of serum overnight. Subsequently, cells were incubated with 5 mM L-Alanine and 5mM [U-13C]-L-Alanine in serum-starved medium (DMEM/0.5% serum). Additionally, fresh media containing L-Alanine and [U-13C]-L-Alanine were exchanged 2 h before metabolite extraction for metabolic analysis."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Cells were homogenized with the first solvent (the mixture of chloroform, methanol and water (1:2:1, v/v/v)) for 30 s at 4 °C and then centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant was collected and deposit was re-homogenized with the second solvent (methanol alone) before a second centrifugation. The 2 supernatants were mixed, and aliquot of sample was transferred to a GC sampling vial containing 5 μL 0.1 mg/mL ribitol (Sigma) as an analytical internal standard and then dried in a vacuum centrifuge concentrator before the subsequent derivatization. A total of 2 technical replicates were prepared for each sample."},

"CHROMATOGRAPHY":{"INSTRUMENT_NAME":"Thermo Scientific Trace GC Ultra with DSQ II GC/MS","COLUMN_NAME":"Agilent DB5-MS (30m x 0.25mm, 0.25um)","COLUMN_TEMPERATURE":"270 °C","FLOW_GRADIENT":"none","FLOW_RATE":"1.0 mL/min","SOLVENT_A":"none","SOLVENT_B":"none","CHROMATOGRAPHY_TYPE":"GC"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Scientific Trace GC Ultra with DSQ II GC/MS","INSTRUMENT_TYPE":"Triple quadrupole","MS_TYPE":"EI","MS_COMMENTS":"samples was derivatized and then used to firstly protect carbonyl moieties through methoximation, through a 90 min 37 ℃ reaction with 40 μL of 20 mg/mL methoxyamine hydrochloride (Sigma-Aldrich) in pyridine, followed by derivatization of acidic protons through a 30 min 37 0C reaction with the addition of 80 μL N-methyl-N-trimethylsilyltrifluoroace-tamide (MSTFA, Sigma-Aldrich). The derivatized sample of 1 μL was injected into a 30m × 250 μm i.d. × 0.25 μm DBS-MS column using splitless injection and analysis was carried out by Trace DSQ II (Thermo Scientific). The initial temperature of the GC oven was held at 85 0C for 5 min followed by an increase to 330 0C at a rate of 15 0C min-1 then held for 5 min. Helium was used as carrier gas and flow was kept constant at 1 mL min-1. The MS was operated in a range of 50-600 m/z.","ION_MODE":"POSITIVE","MS_RESULTS_FILE":"ST002877_AN004714_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes"}

}