Project description:In the development of a desorption electrospray ionization (DESI) workflow for spatial metabolomics, we investigated the impact of two commonly used solvent systems, 90% acetonitrile (ACN) and 90% methanol (MeOH), on the spatial metabolomic profiling of multiple murine tissues. The performance of both solvents was evaluated across several metabolite classes (central carbon metabolites, amino acids, and fatty acids). While the ACN-based solvent system led to higher signal intensities of small polar metabolites involved in glycolysis, the tricarboxylic acid (TCA) cycle, and amino acid metabolism, the MeOH-based solvent system provided superior signal intensities of fatty acids. These findings demonstrate that solvent composition differentially influences metabolite extraction and ionization processes in DESI and should be carefully matched to the biological question and metabolite classes of interest. To illustrate the utility of the optimized workflow, the ACN solvent system was applied to a rat model of renal ischemic injury. Spatial mapping of metabolites across distinct kidney regions (cortex, outer medulla, and inner medulla) revealed pronounced region-specific metabolic changes between normoxic and ischemic conditions. Together, these results demonstrate the importance of solvent selection in DESI-based spatial metabolomics and showcase the ability of this approach to uncover spatially resolved metabolic adaptations associated with tissue injury.
Project description:During senescence of detached rice leaves, tryptophan (Trp) and Trp-derived secondary metabolites such as serotonin and 4-coumaroylserotonin accumulated in concert with methanol (MeOH) production. This senescence-induced MeOH induction was closely associated with levels of pectin methylesterase (PME)1 mRNA and PME enzyme activity. Exogenous challenge of detached rice leaves with 1% MeOH accelerated Trp and serotonin biosynthesis with induction of the corresponding genes. No other solvents including ethanol resulted in a Trp-inducing effect. This MeOH-induced Trp synthesis was positively regulated by abscisic acid but negatively regulated by cytokinin, suggesting hormonal involvement on the action of MeOH. Endogenous overproduction or suppression of MeOH either by PME1 overexpression or RNAi gene silencing revealed that PME1 overexpressing lines produced twofold higher Trp levels with elevated Trp biosynthetic gene expression, whereas RNAi lines showed twofold reduction in Trp level in healthy control rice leaves, suggesting that MeOH acts as an endogenous elicitor to enhance Trp biosynthesis. Among many transcription factors induced following MeOH treatment, the WRKY family showed significant induction patterns of which WRKY14 appeared to play a key regulatory role in MeOH-induced Trp and Trp-derived secondary metabolite biosynthesis. Total RNAs were extracted from the detached rice leaves with 1% MeOH or distilled water for 1 d, and gene expression was compared between the two groups with two replicates. DW, detached leaves in distilled water for 1 day; MeOH (2-replications), methanol treated detached leaves at the same time point as control. 2 sets of separately normalized data; DW-MeOH(1) and MeOH(2).
Project description:Use CEN quechers method to extract 2.5g honey and use PSA to clean up matrix. Using ACN-water-0.01%HCOOH for compound separation and HRMS analysis with Thermo Orbitrap Exploris 120. Data was acquired in Full scan-ddms2 mode. This included a full scan over the m/z range 100- 1000 at full width at half maximum (FWHM) resolution of 60,000, and a data-dependent-MS2 scan at FWHM resolution of 15,000 on the top 4 ions. The ionization was performed in positive ESI with an inlusion list collated from OPPIN website, and to gain more information about fragment ions in the QC sample, we use an Automated Exclusion List Generation workflow, so one QC sample finally gave 3 injections.
Project description:In order to get mechanistic insights into the cardiomyogenic and differentiation-promoting activity of Crataegus spp. Extract WS®1442 and the identified bioactive subfractions (MeOH eluate), we have employed whole genome microarray expression profiling after 6 hours and 24 hours of treatment. We found that stress-associated genes are affected as well as specific signaling pathways such as TGFbeta, FGF, BDNF and retinoic acid.
Project description:During senescence of detached rice leaves, tryptophan (Trp) and Trp-derived secondary metabolites such as serotonin and 4-coumaroylserotonin accumulated in concert with methanol (MeOH) production. This senescence-induced MeOH induction was closely associated with levels of pectin methylesterase (PME)1 mRNA and PME enzyme activity. Exogenous challenge of detached rice leaves with 1% MeOH accelerated Trp and serotonin biosynthesis with induction of the corresponding genes. No other solvents including ethanol resulted in a Trp-inducing effect. This MeOH-induced Trp synthesis was positively regulated by abscisic acid but negatively regulated by cytokinin, suggesting hormonal involvement on the action of MeOH. Endogenous overproduction or suppression of MeOH either by PME1 overexpression or RNAi gene silencing revealed that PME1 overexpressing lines produced twofold higher Trp levels with elevated Trp biosynthetic gene expression, whereas RNAi lines showed twofold reduction in Trp level in healthy control rice leaves, suggesting that MeOH acts as an endogenous elicitor to enhance Trp biosynthesis. Among many transcription factors induced following MeOH treatment, the WRKY family showed significant induction patterns of which WRKY14 appeared to play a key regulatory role in MeOH-induced Trp and Trp-derived secondary metabolite biosynthesis.
Project description:Transcriptomics study which main goal is to elucidate the programme of gene expression triggered by water stress in leaflets of the drought-tolerant wild-related tomato Solanum pennellii (acc. PE47) compared with domesticated tomato (S. lycopersicum, cv. P73). In this study we used S. lycopersicum (Sl) (cv. P73) and S. pennellii (Sp) (acc. PE47) species displaying remarkable divergences regarding drought tolerance, to investigate the physiological and molecular responses in leaves of plants grown without stress (control) and after four days of water withholding (water stress, WS), when plant water loss was significant but leaves did not show visual dehydration symptoms yet. Significant physiological differences between species were found, showing Sp leaves higher ability to avoid water loss. Leaf transcriptomic analysis showed important constitutive expression differences between Sp and Sl, including genes with unknown function. In relation to the genes specifically induced by drought in Sp, those linked to stomatal closure, cell wall and primary carbohydrate metabolism and, specially, nitrogen metabolism were identified. Thus, genes linked to NH4+ assimilation, GOGAT/GS cycle and the GDH- and GABA-shunt were specifically induced by water stress in leaves of Sp. Our results showed also the up-regulation in Sp of genes involved in JA biosynthesis pathway, which were induced in both conditions, whereas genes involved in ET biosynthesis were specifically induced under WS. Regarding ET signaling, ERF genes were up-regulated by WS in Sp, hinting at the importance of these transcriptional regulators in the drought response of Sp.
Project description:New mechanisms-of-action of anthocyanins (ACNs) provided by a red-fleshed apple compared with a white-fleshed apple ACN-poor, and with an ACN-rich extract on the proteome profile of aorta and heart as cardiovascular key tissues were determined. Hypercholesterolemic Wistar rats were separated into the corresponding groups to analyze the proteomic profile of the aorta and heart tissues using nano-liquid chromatography coupled to mass-spectrometry. Red-fleshed apple downregulated CRP, C1QB and CFP related-inflammation. White-fleshed apple reduced C1QB, CFB, CFD, C3, and C9 related to the complement system, reduced MB and CP related to iron metabolism, and increased ME1, PKM, and PC related to energy homeostasis. ACN-rich extract increased FMOD, TAGLN, and CAP1 related to cellular structure and decreased PRKACA, IQGAP1, and HSP90AB1 related to cellular signaling. Red-fleshed apple rich in ACNs suggested an anti-inflammatory effect while white-fleshed apple reduced the complement system protein-related. An apple matrix effect reduced inflammatory proteins regardless their ACN content.
Project description:Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH into toxic formaldehyde (FA). Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and the modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) in volunteers after pectin intake showed various responses for 30 differentially regulated mRNAs. Most of the mRNAs were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes did not show significant change. A qRT-PCR analysis of volunteer WBC after pectin and red wine intake confirmed the complicated dependence between plasma MeOH content and the mRNA accumulation of previously identified genes, namely GAPDH and SNX27, and MME, SORL1, DDIT4, HBA and HBB genes revealed in this study. We hypothesized that human plasma MeOH, which is replenished from endogenous and exogenous sources (diet), has an impact on the WBC mRNA levels of genes involved in AD pathogenesis and signaling.
Project description:Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH into toxic formaldehyde (FA). Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and the modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) in volunteers after pectin intake showed various responses for 30 differentially regulated mRNAs. Most of the mRNAs were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes did not show significant change. A qRT-PCR analysis of volunteer WBC after pectin and red wine intake confirmed the complicated dependence between plasma MeOH content and the mRNA accumulation of previously identified genes, namely GAPDH and SNX27, and MME, SORL1, DDIT4, HBA and HBB genes revealed in this study. We hypothesized that human plasma MeOH, which is replenished from endogenous and exogenous sources (diet), has an impact on the WBC mRNA levels of genes involved in AD pathogenesis and signaling.
Project description:Methanol (MeOH) is considered to be a poison in humans because of the alcohol dehydrogenase (ADH)-mediated conversion of MeOH into toxic formaldehyde (FA). Our recent genome-wide analysis of the mouse brain demonstrated that an increase in endogenous MeOH after ADH inhibition led to a significant increase in the plasma MeOH concentration and the modification of mRNA synthesis. These findings suggest endogenous MeOH involvement in homeostasis regulation by controlling mRNA levels. Here, we demonstrate directly that study volunteers displayed increasing concentrations of MeOH and FA in their blood plasma when consuming citrus pectin, ethanol and red wine. A microarray analysis of white blood cells (WBC) in volunteers after pectin intake showed various responses for 30 differentially regulated mRNAs. Most of the mRNAs were somehow involved in the pathogenesis of Alzheimer's disease (AD). There was also a decreased synthesis of hemoglobin mRNA, HBA and HBB, the presence of which in WBC RNA was not a result of red blood cells contamination because erythrocyte-specific marker genes did not show significant change. A qRT-PCR analysis of volunteer WBC after pectin and red wine intake confirmed the complicated dependence between plasma MeOH content and the mRNA accumulation of previously identified genes, namely GAPDH and SNX27, and MME, SORL1, DDIT4, HBA and HBB genes revealed in this study. We hypothesized that human plasma MeOH, which is replenished from endogenous and exogenous sources (diet), has an impact on the WBC mRNA levels of genes involved in AD pathogenesis and signaling.