#METABOLOMICS WORKBENCH Fightingkyb_2024_20240820_064258 DATATRACK_ID:5128 STUDY_ID:ST003425 ANALYSIS_ID:AN005624 PROJECT_ID:PR002117
VERSION             	1
CREATED_ON             	August 21, 2024, 11:28 pm
#PROJECT
PR:PROJECT_TITLE                 	A mouse protozoan boosts antigen-specific mucosal IgA responses in a specific
PR:PROJECT_TITLE                 	lipid metabolism- and signaling-dependent manner
PR:PROJECT_SUMMARY               	IgA antibodies play an important role in mucosal immunity. However, there is
PR:PROJECT_SUMMARY               	still no effective way to consistently boost mucosal IgA responses, and the
PR:PROJECT_SUMMARY               	factors influencing these responses are not fully understood. We observed that
PR:PROJECT_SUMMARY               	colonization with the murine intestinal symbiotic protozoan Tritrichomonas
PR:PROJECT_SUMMARY               	musculis (T.mu) boosted antigen-specific mucosal IgA responses in wild-type
PR:PROJECT_SUMMARY               	C57BL/6 mice. This enhancement was attributed to the accumulation of free
PR:PROJECT_SUMMARY               	arachidonic acid (ARA) in the intestinal lumen, which served as a signal to
PR:PROJECT_SUMMARY               	stimulate the production of antigen-specific mucosal IgA. When ARA was prevented
PR:PROJECT_SUMMARY               	from undergoing its downstream metabolic transformation using the 5-lipoxygenase
PR:PROJECT_SUMMARY               	inhibitor zileuton or by blocking its downstream biological signaling through
PR:PROJECT_SUMMARY               	genetic deletion of the Leukotriene B4 receptor 1 (Blt1), the T.mu-mediated
PR:PROJECT_SUMMARY               	enhancement of antigen-specific mucosal IgA production was suppressed. Moreover,
PR:PROJECT_SUMMARY               	both T.mu transfer and dietary supplementation of ARA augmented the efficacy of
PR:PROJECT_SUMMARY               	an oral vaccine against Salmonella infection, with this effect being dependent
PR:PROJECT_SUMMARY               	on Blt1. Our findings elucidate a tripartite circuit linking nutrients from the
PR:PROJECT_SUMMARY               	diet or intestinal microbiota, host lipid metabolism, and the mucosal humoral
PR:PROJECT_SUMMARY               	immune response.
PR:INSTITUTE                     	Xuzhou medical university
PR:LAST_NAME                     	Kou
PR:FIRST_NAME                    	Yanbo
PR:ADDRESS                       	Tongshan road 209, Xuzhou, Jiangsu, 221004, China
PR:EMAIL                         	fightingkyb@163.com
PR:PHONE                         	+86-051683262123
#STUDY
ST:STUDY_TITLE                   	Targeted free fatty acids metabolomics studies on serum, cecal content, cecum
ST:STUDY_TITLE                   	tissue, chow diet, and T.mu cells.
ST:STUDY_SUMMARY                 	Serum, cecal content, cecum tissue samples of murine, chow diet, and T.mu cells
ST:STUDY_SUMMARY                 	were collected to perform the targeted free fatty acids metabolome analysis. The
ST:STUDY_SUMMARY                 	aim of this study was to verify that whether T.mu could release free PUFA
ST:STUDY_SUMMARY                 	(especially ARA) to the intestinal tract of its host by comparing the PUFA
ST:STUDY_SUMMARY                 	concentration in the freshly isolated T.mu cells, chow diet, cecal content and
ST:STUDY_SUMMARY                 	serum of the host (Mus musculus).Based on the result of this targeted free fatty
ST:STUDY_SUMMARY                 	acids metabolomics studies, we found that T.mu could release ARA to the
ST:STUDY_SUMMARY                 	intestinal tract of its host and increase the concentration of ARA in its host's
ST:STUDY_SUMMARY                 	intestinal tract.
ST:INSTITUTE                     	Xuzhou medical university
ST:LAST_NAME                     	Kou
ST:FIRST_NAME                    	Yanbo
ST:ADDRESS                       	Tongshan road 209, Xuzhou, Jiangsu, 221004, China
ST:EMAIL                         	fightingkyb@163.com
ST:PHONE                         	+86-051683262123
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENDER                        	Not applicable
#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           	-	T_mu	Sample source:T_mu cells | Treatment:isolated from cecal content	RAW_FILE_NAME(Raw file name)=T_mu.CDF
SUBJECT_SAMPLE_FACTORS           	-	Diet	Sample source:chow diet | Treatment:ground to power	RAW_FILE_NAME(Raw file name)=Diet.CDF
SUBJECT_SAMPLE_FACTORS           	-	Ctrl_serum	Sample source:serum | Treatment:control	RAW_FILE_NAME(Raw file name)=Ctrl_serum.CDF
SUBJECT_SAMPLE_FACTORS           	-	T_mu_serum	Sample source:serum | Treatment:colonized with Tmu	RAW_FILE_NAME(Raw file name)=Ctrl_serum.CDF
SUBJECT_SAMPLE_FACTORS           	-	Ctrl_cecum	Sample source:cecum tissue | Treatment:control	RAW_FILE_NAME(Raw file name)=Ctrl_serum.CDF
SUBJECT_SAMPLE_FACTORS           	-	T_mu_cecum	Sample source:cecum tissue | Treatment:colonized with Tmu	RAW_FILE_NAME(Raw file name)=Ctrl_serum.CDF
SUBJECT_SAMPLE_FACTORS           	-	Ctrl_content	Sample source:cecal content | Treatment:control	RAW_FILE_NAME(Raw file name)=Ctrl_serum.CDF
SUBJECT_SAMPLE_FACTORS           	-	T_mu_content	Sample source:cecal content | Treatment:colonized with Tmu	RAW_FILE_NAME(Raw file name)=Ctrl_serum.CDF
#COLLECTION
CO:COLLECTION_SUMMARY            	WT C57/BL6 mice were colonized with or without T.mu for 7 days, and the serum,
CO:COLLECTION_SUMMARY            	cecum tissue, cecal content samples were collected on day 7. For T.mu cells
CO:COLLECTION_SUMMARY            	sample, T.mu cells were isolated and counted from T.mu positive cecal content.
CO:COLLECTION_SUMMARY            	Samples were washed with cold PBS and then flash-frozen in liquid nitrogen.
CO:SAMPLE_TYPE                   	Serum, cecum tissue, cecal content, T.mu cells, chow diet
#TREATMENT
TR:TREATMENT_SUMMARY             	WT C57/BL6 mice were colonized with or without T.mu for 7 days, and the serum,
TR:TREATMENT_SUMMARY             	cecum tissue, cecal content samples were collected on day 7. For T.mu cells
TR:TREATMENT_SUMMARY             	sample, T.mu cells were isolated and counted from T.mu positive cecal content.
TR:TREATMENT_SUMMARY             	For diet sample, the chow diet power was used.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	The weighed cecal content, cecum tissue, serum, animal chow diet powder, or
SP:SAMPLEPREP_SUMMARY            	enumerated T.mu cells were transferred into new 2 mL EP tubes followed
SP:SAMPLEPREP_SUMMARY            	extraction with 500 μL extracting solution [Isopropanol : n-Hexane = 2:3
SP:SAMPLEPREP_SUMMARY            	(V:V)], including 0.2 mg/L internal standard). After homogenized in a ball mill
SP:SAMPLEPREP_SUMMARY            	for 4 min at 40 Hz and a 5 min ultrasound treatment, the samples were
SP:SAMPLEPREP_SUMMARY            	centrifuged 16200 × g for 15 min at 4 °C. Then the supernatants were
SP:SAMPLEPREP_SUMMARY            	transferred into new 2 mL EP tubes followed with nitrogen blow dry. The dried
SP:SAMPLEPREP_SUMMARY            	samples were resuspended in 500 μL of methanol: trimethylsilyl diazomethane
SP:SAMPLEPREP_SUMMARY            	solution (1:2), after standing 30 min at room temperature, another nitrogen blow
SP:SAMPLEPREP_SUMMARY            	dry was performed. Then, the samples were resuspended in 160 μL of n-hexane
SP:SAMPLEPREP_SUMMARY            	followed with a centrifugation of 16200 × g for 1 min. Finally, the
SP:SAMPLEPREP_SUMMARY            	supernatants were transferred into new vials for GC-MS analysis.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	DB-FastFAME capillary column (90 m × 0.25 mm × 0.25 µm, Agilent Technologies)
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890B
CH:COLUMN_NAME                   	Agilent DB-FastFAME capillary column (90 m × 0.25 mm × 0.25 um)
CH:SOLVENT_A                     	-
CH:SOLVENT_B                     	-
CH:FLOW_GRADIENT                 	-
CH:FLOW_RATE                     	-
CH:COLUMN_TEMPERATURE            	230
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 5977B
MS:INSTRUMENT_TYPE               	Single quadrupole
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Mass spectrometry data were acquired in Scan/SIM mode over an m/z range of
MS:MS_COMMENTS                   	33-400 after a solvent delay of 7 minutes. Fatty acid methyl esters (FAMEs) were
MS:MS_COMMENTS                   	identified by comparing them to commercial FAME standards (ANPEL, Shanghai,
MS:MS_COMMENTS                   	China, Cat# CDAA-252). The absolute concentrations of FAMEs were calculated
MS:MS_COMMENTS                   	using calibration curves based on the internal standard method.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	μg/ml for serum, μg/g for cecal content, μg/g for diet, μg/8*10^7 T.mu cells
MS_METABOLITE_DATA_START
Samples	T_mu_content	Ctrl_content	Ctrl_cecum	T_mu_cecum	Ctrl_serum	T_mu_serum	T_mu	Diet
Factors	Sample source:cecal content | Treatment:colonized with Tmu	Sample source:cecal content | Treatment:control	Sample source:cecum tissue | Treatment:control	Sample source:cecum tissue | Treatment:colonized with Tmu	Sample source:serum | Treatment:control	Sample source:serum | Treatment:colonized with Tmu	Sample source:T_mu cells | Treatment:isolated from cecal content	Sample source:chow diet | Treatment:ground to power
Oleic acid	37627.95	56029.43	30279.12	60759.18	45231.54	32806.51	19580.48	466521.47
Palmitic Acid	75445.56	67998.47	34576.42	53400.25	80887	59882.24	95952.18	529736.32
Octanoic Acid	27.6	5.54	0	0	40.56	31.08	36.32	1835.27
Decanoic Acid	54.17	25.38	39.07	29.52	31.14	36.76	3506	49.82
Elaidic acid	0	0	0	0	81.73	41.62	4192.23	0
Lauric Acid	443.29	20.22	86.38	91.78	13.72	5.14	91.26	642.04
Linoleic acid	69682.13	69057.69	23288.7	50446.44	70355.06	64783.42	66078.22	1361381.6
Myristic Acid	2204.33	1819.72	650.5	1825.61	1181.47	599.61	1548.93	25005.82
Pentadecanoic Acid	3562.1	1144.14	451.22	686.58	434.63	281.83	4213.02	4075.64
Stearic Acid	66344.88	38976.5	29705.75	30269.88	35981.51	30912.09	45962	204561.27
Tridecylic Acid	237.73	28.09	4.66	34.19	0	0	36.8	152.76
Behenic Acid	8345.2	5311.53	393.16	609.45	632.88	345.05	2157.86	18918.39
Undecanoic Acid	0	0	6.01	0.23	0	0	12.18	13.12
all-cis-5,8,11,14-Eicosatetraenoic Acid	16445.13	3779.78	19633.23	21338.07	18269.15	16387.36	13631.63	6265.68
Tricosanoic Acid	1534.17	1214.7	71.96	63.71	0	0	288.27	1558.46
Linolenic acid	3233.24	4060.03	548.59	1734.04	2401.3	2538.86	3067.46	254061.25
all-cis-4,7,10,13,16-Docosapentaenoic Acid	195.99	93.67	516.03	503.7	548.96	438.07	282.89	0
all-cis-5,8,11,14,17-Eicosapentaenoic Acid	1057.51	521.05	959.08	778.96	1052.08	878.15	1197.67	24629.76
Myristoleic acid	1311.1	150.44	35.96	60.21	107.44	58.96	232.32	1000.81
Lignoceric Acid	6128.87	4398.34	314.45	362.33	59.37	45.42	708.52	5689.14
Erucic acid	2234.25	844.42	178.27	421.8	122.32	130.02	476.68	2279.46
cis-6-Octadecenoic acid	0	0	0	0	0	0	2976.87	0
all-cis-4,7,10,13,16,19-Docosahexaenoic Acid	6828.01	1291.61	6227.32	7617.97	17286.6	14454.94	6893.52	17846.87
Arachidic Acid	6570.67	5039.45	372.68	455.85	111.98	86.35	1320.62	11317
all-cis-7,10,13,16-Docosatetraenoic Acid	942.63	303.98	1978.27	2439.37	337.17	410.5	394.25	879.1
cis-11-Eicosenoic Acid	3492.32	2759.65	526.05	1602.77	1415.14	872.79	851.33	29203.73
Heptadecanoic Acid	2452.18	1665.55	893.6	855.64	803.85	630.46	2490.08	2642.19
Heneicosanoic Acid	1026.04	962.22	48.68	82.18	22.29	10.25	241.63	1065.95
15-Nervonic acid	2031.72	804.39	0	0	0	0	0	6152.66
cis,cis,cis-8,11,14-Linolenic Acid	2650.28	1001.68	2993.7	3405.89	4047.14	2135.56	1534.45	1163.22
gamma-Linolenic acid	205.71	134.08	238.15	383.13	725.47	917.38	84.89	1236.35
Palmitoleic acid	3071.94	3714.46	2444.58	8255.91	8680.01	3255.93	1491.37	62498.55
trans-11-Octadecenoic acid	49515.3	37244.25	1231.04	3722.62	283.07	41	48051.64	0
cis,cis-11,14-Eicosadienoic Acid	861.62	408.81	1005.83	1128.37	1084.43	561.85	296.75	3764.44
Linoelaidic acid	22.18	115.66	0	0	0	0	118.66	454.48
all-cis-7,10,13,16,19-Docosapentaenoic acid	461.11	234.58	1048.62	1224.34	917.42	674.85	257.9	3799.36
Palmitelaidic acid	0	0	67.69	1356.86	0	0	0	4794.52
cis-10-Heptadecenoic Acid	0	0	30.87	349.12	0	0	0	0
all-cis-11,14,17-Eicosatrienoic Acid	0	0	38.3	44.67	54.19	41	148.8	375.3
cis,cis-13,16-Docosadienoic Acid	0	0	0	0	0	0	0	85.38
Myristelaidic acid	0	0	0	0	0	0	0	170.45
cis-11-Octadecenoic acid	9206.95	6149.43	6730.93	9885.31	7274.64	3609.61	3059.87	96145.78
trans-10-Heptadecenoic acid	52.3	23.25	91.48	84.6	64.35	61.48	62.32	0
trans-13-Docosenoic acid	295.9	278.47	64.15	72.98	93.81	46.44	296.2	3967.64
6-Octadecenoic acid	0	0	0	0	0	0	0	0
cis-10-Pentadecenoic Acid	342.16	235.57	0	0	0	0	291.24	0
trans-10-pentadecenoic acid	0	0	0	0	0	0	0	0
trans-10-Nonadecenoic acid	401.22	444.27	0	0	62.2	79.64	295.49	0
trans-11-Eicosenoic acid	182.12	111.14	0	0	0	0	0	0
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CAS	Molecular weight	KEGG ID
Oleic acid	112-80-1	282.4614	C00712
Palmitic Acid	57-10-3	256.4241	C00249
Octanoic Acid	124-07-2	144.21	C06423
Decanoic Acid	334-48-5	172.26	C01571
Elaidic acid	112-79-8	282.4614	C01712
Lauric Acid	143-07-7	200.32	C02679
Linoleic acid	60-33-3	280.4455	C01595
Myristic Acid	544-63-8	228.37	C06424
Pentadecanoic Acid	1002-84-2	242.4	C16537
Stearic Acid	57-11-4	284.4772	C01530
Tridecylic Acid	638-53-9	214.34	C17076
Behenic Acid	112-85-6	340.5836	C08281
Undecanoic Acid	112-37-8	186.291	C17715
all-cis-5,8,11,14-Eicosatetraenoic Acid	506-32-1	304.4669	C00219
Tricosanoic Acid	2433-96-7	354.61	NA
Linolenic acid	463-40-1	278.4296	C06427
all-cis-4,7,10,13,16-Docosapentaenoic Acid	25182-74-5	330.5	C16513
all-cis-5,8,11,14,17-Eicosapentaenoic Acid	10417-94-4	302.451	C06428
Myristoleic acid	544-64-9	226.355	C08322
Lignoceric Acid	557-59-5	368.6367	C08320
Erucic acid	112-86-7	338.5677	C08316
cis-6-Octadecenoic acid	593-39-5	282.4614	C08363
all-cis-4,7,10,13,16,19-Docosahexaenoic Acid	6217-54-5	328.4883	C06429
Arachidic Acid	506-30-9	312.5304	C06425
all-cis-7,10,13,16-Docosatetraenoic Acid	28874-58-0	332.52	C16527
cis-11-Eicosenoic Acid	5561-99-9	310.5145	C16526
Heptadecanoic Acid	506-12-7	270.45	NA
Heneicosanoic Acid	2363-71-5	326.56	NA
15-Nervonic acid	506-37-6	366.6208	C08323
cis,cis,cis-8,11,14-Linolenic Acid	1783-84-2	306.4828	C03242
gamma-Linolenic acid	506-26-3	278.4296	C06426
Palmitoleic acid	373-49-9	254.4082	C08362
trans-11-Octadecenoic acid	693-72-1	282.4614	C08367
cis,cis-11,14-Eicosadienoic Acid	5598-38-9	308.4986	C16525
Linoelaidic acid	506-21-8	280.4455	NA
all-cis-7,10,13,16,19-Docosapentaenoic acid	24880-45-3	330.5042	C16513
Palmitelaidic acid	10030-74-7	254.4082	NA
cis-10-Heptadecenoic Acid	29743-97-3	268.4348	NA
all-cis-11,14,17-Eicosatrienoic Acid	17046-59-2	306.4828	C16522
cis,cis-13,16-Docosadienoic Acid	17735-98-7	336.5518	C16533
Myristelaidic acid	50286-30-1	226.36	NA
cis-11-Octadecenoic acid	506-17-2 	282.46	C21944
trans-10-Heptadecenoic acid	126761-43-1	268.4348	NA
trans-13-Docosenoic acid	506-33-2	338.57	NA
6-Octadecenoic acid	593-40-8 	282.46	NA
cis-10-Pentadecenoic Acid	84743-29-3	240.38	NA
trans-10-pentadecenoic acid	321744-58-5	240.38	NA
trans-10-Nonadecenoic acid	67228-95-9	296.5	NA
trans-11-Eicosenoic acid	62322-84-3	310.51	NA
METABOLITES_END
#END