MetaboLights

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ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/m_MTBLS14563_GC-MS_alternating_high-polarity_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/a_MTBLS14563_GC-MS_alternating_high-polarity.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/s_MTBLS14563.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsEGFP-2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsHMC-8.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsHMC-6.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsEGFP-4.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsHMC-2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/QC1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsHMC-3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsEGFP-7.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsHMC-5.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/QC-2.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsEGFP-3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsEGFP-5.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsHMC-7.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsHMC-1.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsEGFP-8.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsEGFP-6.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsHMC-4.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/QC-3.rawftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563/FILES/RAW_FILES/dsEGFP-1.rawprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS14563 Principal component analysis (PCA), orthogonal projections to latent structures discriminant analysis (OPLS-DA) score scatter plot, clustering heatmap analysis, and pathway analysis were performed using online software MetaboAnalyst (http://www.metaboanalyst.ca/). Identification of differentially abundant metabolites was performed following a workflow as previously study [26], whereby p-values were calculated with two-tailed Student’s t-tests and a Wilcoxon rank-sum test, followed by a false discovery rate (FDR) adjustment [27]. After validation using multiple testing corrections, the differential abundance of metabolites with p < 0.05 and fold change (FC) > 2 were chosen for further pathway analysis. Z-score analysis scaled each metabolite according to a reference distribution, calculated based on the mean and standard deviation of the control.MetaboLightsPublicGas Chromatography MS - alternating - high-polarity Samples were analyzed by gas chromatography coupled with mass spectrometry (GC-MS) on an Agilent 7890B gas chromatography-time-of-flight MS system equipped with an Agilent DB-5MS capillary column (30 m × 250 μm × 0.25 μm), using helium gas at a flow rate of 1.0 mL/min. Inlet and transmission-line temperatures were maintained at 270°C, with an ion source temperature of 220°C. The heating procedure involved 80°C (2 min), 80–300°C (12°C/min), and 300°C (8 min). Full scan mode detection (m/z 50–500) was performed at an ionization voltage of 70 eV, with a scan rate of 25 spectra/sec.Hemocyanin negatively regulates phenoloxidase activation via phenylalanine-tyrosine metabolism to enhance immune defense in AHPND-infected Penaeus vannamei.Yonghui Kongshantou universityhepatopancreasmass spectrometry assay To profile shrimp hepatopancreas metabolites, 30 mg hepatopancreas samples were mixed with pre-cooled methanol/chloroform (v/v = 3/1) and a solution containing 10 μL of an internal standard. After vortexing, the mixture was centrifuged at 14,000 g for 20 min at 4°C. The supernatant was collected for vacuum drying, then each sample was treated with 50 μL methoxyamine salt reagent (dissolved in 20 mg/mL pyridine), mixed, and derivatized at 30°C for 2 h. Samples were then further derived by adding 50 μL MSTFA (containing 1% TMCS, v/v), incubated at 37.5°C for 1 h, then thawed in an ice bath before being mixed with pre-cooled methanol/chloroform (v/v = 3/1) and the internal standard (10 μL).Penaeus vannameihttps://www.ebi.ac.uk/metabolights/MTBLS14563Yonghui Kong. Shantou University. kerwinkon728@gmail.com. Raw data were processed using ChromaTOF software (V4.71, LECO) for baseline denoising and smoothing, peak extraction, deconvolution, and peak alignment. Compounds were identified by matching the FAMEs retention index and fragment ion profiles of metabolites in the local database (including self-built 1000 + standard library and LECO-Fiehn database).ControlRNA interferencekerwinkon728@gmail.comHepatopancreatic cells were prepared by gentle mincing of the hepatopancreas in 1 ml of 0.01 M PBS (pH 7.2) with a sterile pestle MetabolomicsMetabolomicsLECO Pegasus BT TOFMSshrimpphenylalanineAgilent 7890B GCuntargeted analysisHepatopancreasPenaeus vannameityrosineexperimental sampledata-independent acquisitionMetabolomicsLECO Pegasus BT TOFMSshrimpAgilent 7890B GCphenylalanineuntargeted analysisHepatopancreasPenaeus vannameityrosineexperimental sampledata-independent acquisitionSamples were analyzed by gas chromatography coupled with mass spectrometry (GC-MS) on an Agilent 7890B gas chromatography-time-of-flight MS system equipped with an Agilent DB-5MS capillary column (30 m × 250 μm × 0.25 μm), using helium gas at a flow rate of 1.0 mL/min. Inlet and transmission-line temperatures were maintained at 270°C, with an ion source temperature of 220°C. The heating procedure involved 80°C (2 min), 80–300°C (12°C/min), and 300°C (8 min). Full scan mode detection (m/z 50–500) was performed at an ionization voltage of 70 eV, with a scan rate of 25 spectra/sec.falseHemocyanin negatively regulates phenoloxidase activation via phenylalanine-tyrosine metabolism to enhance immune defense in AHPND-infected Penaeus vannameiHemocyanins function not only as oxygen carriers in the hemolymph of arthropods and molluscs but also as an important multifunctional immune molecule. In this study, we observed a significant increase in hemocyanin expression and a decrease in PO activity in Penaeus vannamei infected with acute hepatopancreatic necrosis disease (AHPND). Using gas chromatography-mass spectrometry (GC-MS)-based metabolomics, we found that knockdown of the hemocyanin gene (PvHMC) led to significant reductions in phenylalanine and tyrosine levels. Subsequent qPCR, PO activity assays, and LC-MS analyses revealed that PvHMC knockdown significantly elevated the transcript levels of key enzymes in the phenylalanine-tyrosine metabolic pathway, accompanied by increased L-Dopa levels and enhanced PO activity. In contrast, exogenous supplementation of phenylalanine or tyrosine downregulated these four enzymes and reduced PO activity. Furthermore, after PvHMC knockdown, inhibiting phenylalanine hydroxylase with 4-Chloro-DL-phenylalanine (PCPA) or tyrosine hydroxylase with 3-Iodo-L-tyrosine (3-IT) increased phenylalanine or tyrosine levels, decreased the expression of the corresponding enzymes, and reduced both L-Dopa content and PO activity. Collectively, these results demonstrate that during Vibrio parahaemolyticus (VpAHPND) infection, PvHMC is significantly downregulated and negatively regulates PO activity via the phenylalanine-tyrosine metabolism, thereby enhancing immune defense. This study elucidates a novel metabolic mechanism through underlying PvHMC-mediated regulation of the PO cascade regulation, providing new insights into the interplay between metabolism and immunity in shrimp.2026-05-222026-05-22MTBLS14563