PubMed

Arch Toxicol. 2023 Jan 22. doi: 10.1007/s00204-022-03439-3. Online ahead of print.ABSTRACTAmongst omics technologies, metabolomics should have particular value in regulatory toxicology as the measurement of the molecular phenotype is the closest to traditional apical endpoints, whilst offering mechanistic insights into the biological perturbations. Despite this, the application of untargeted metabolomics for point-of-departure (POD) derivation via benchmark concentration (BMC) modelling is still a relatively unexplored area. In this study, a high-throughput workflow was applied to derive PODs associated with a chemical exposure by measuring the intracellular metabolome of the HepaRG cell line following treatment with one of four chemicals (aflatoxin B1, benzo[a]pyrene, cyclosporin A, or rotenone), each at seven concentrations (aflatoxin B1, benzo[a]pyrene, cyclosporin A: from 0.2048 μM to 50 μM; rotenone: from 0.04096 to 10 μM) and five sampling time points (2, 6, 12, 24 and 48 h). The study explored three approaches to derive PODs using benchmark concentration modelling applied to single features in the metabolomics datasets or annotated metabolites or lipids: (1) the 1st rank-ordered unannotated feature, (2) the 1st rank-ordered putatively annotated feature (using a recently developed HepaRG-specific library of polar metabolites and lipids), and (3) 25th rank-ordered feature, demonstrating that for three out of four chemical datasets all of these approaches led to relatively consistent BMC values, varying less than tenfold across the methods. In addition, using the 1st rank-ordered unannotated feature it was possible to investigate temporal trends in the datasets, which were shown to be chemical specific. Furthermore, a possible integration of metabolomics-driven POD derivation with the liver steatosis adverse outcome pathway (AOP) was demonstrated. The study highlights that advances in technologies enable application of in vitro metabolomics at scale; however, greater confidence in metabolite identification is required to ensure PODs are mechanistically anchored.PMID:36683062 | DOI:10.1007/s00204-022-03439-3

Anal Bioanal Chem. 2023 Jan 23. doi: 10.1007/s00216-022-04489-3. Online ahead of print.ABSTRACTOxylipins derived from the cyclooxygenase (COX) and lipoxygenase (LOX) pathways of the arachidonic acid (ARA) cascade are essential for the regulation of the inflammatory response and many other physiological functions. Comprehensive analytical methods comprised of oxylipin and protein abundance analysis are required to fully understand mechanisms leading to changes within these pathways. Here, we describe the development of a quantitative multi-omics approach combining liquid chromatography tandem mass spectrometry-based targeted oxylipin metabolomics and proteomics. As the first targeted proteomics method to cover these pathways, it enables the quantitative analysis of all human COX (COX-1 and COX-2) and relevant LOX pathway enzymes (5-LOX, 12-LOX, 15-LOX, 15-LOX-2, and FLAP) in parallel to the analysis of 239 oxylipins with our targeted oxylipin metabolomics method from a single sample. The detailed comparison between MRM3 and classical MRM-based detection in proteomics showed increased selectivity for MRM3, while MRM performed better in terms of sensitivity (LLOQ, 16-122 pM vs. 75-840 pM for the same peptides), linear range (up to 1.5-7.4 μM vs. 4-368 nM), and multiplexing capacities. Thus, the MRM mode was more favorable for this pathway analysis. With this sensitive multi-omics approach, we comprehensively characterized oxylipin and protein patterns in the human monocytic cell line THP-1 and differently polarized primary macrophages. Finally, the quantification of changes in protein and oxylipin levels induced by lipopolysaccharide stimulation and pharmaceutical treatment demonstrates its usefulness to study molecular modes of action involved in the modulation of the ARA cascade.PMID:36683060 | DOI:10.1007/s00216-022-04489-3

J Exp Clin Cancer Res. 2023 Jan 23;42(1):27. doi: 10.1186/s13046-022-02582-0.ABSTRACTBACKGROUND: Pleural mesothelioma (PM) is an aggressive malignancy with poor prognosis. Unlike many other cancers, PM is mostly characterized by inactivation of tumor suppressor genes. Its highly malignant nature in absence of tumor driving oncogene mutations indicates an extrinsic supply of stimulating signals by cells of the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are an abundant cell type of the TME and have been shown to drive the progression of several malignancies. The aim of the current study was to isolate and characterize patient-derived mesothelioma-associated fibroblasts (Meso-CAFs), and evaluate their impact on PM cells.METHODS: Meso-CAFs were isolated from surgical specimens of PM patients and analyzed by array comparative genomic hybridization, next generation sequencing, transcriptomics and proteomics. Human PM cell lines were retrovirally transduced with GFP. The impact of Meso-CAFs on tumor cell growth, migration, as well as the response to small molecule inhibitors, cisplatin and pemetrexed treatment was investigated in 2D and 3D co-culture models by videomicroscopy and automated image analysis.RESULTS: Meso-CAFs show a normal diploid genotype without gene copy number aberrations typical for PM cells. They express CAF markers and lack PM marker expression. Their proteome and secretome profiles clearly differ from normal lung fibroblasts with particularly strong differences in actively secreted proteins. The presence of Meso-CAFs in co-culture resulted in significantly increased proliferation and migration of PM cells. A similar effect on PM cell growth and migration was induced by Meso-CAF-conditioned medium. Inhibition of c-Met with crizotinib, PI3K with LY-2940002 or WNT signaling with WNT-C59 significantly impaired the Meso-CAF-mediated growth stimulation of PM cells in co-culture at concentrations not affecting the PM cells alone. Meso-CAFs did not provide protection of PM cells against cisplatin but showed significant protection against the EGFR inhibitor erlotinib.CONCLUSIONS: Our study provides the first characterization of human patient-derived Meso-CAFs and demonstrates a strong impact of Meso-CAFs on PM cell growth and migration, two key characteristics of PM aggressiveness, indicating a major role of Meso-CAFs in driving PM progression. Moreover, we identify signaling pathways required for Meso-CAF-mediated growth stimulation. These data could be relevant for novel therapeutic strategies against PM.PMID:36683050 | DOI:10.1186/s13046-022-02582-0

Environ Pollut. 2023 Jan 19:121094. doi: 10.1016/j.envpol.2023.121094. Online ahead of print.ABSTRACTOver the last decade, significant effort has been made to understand phytohormonal functions (e.g., cytokinins (CKs) and abscisic acid (ABA)) in metal stress responses of higher plants and algae. Despite the potential for these phytohormones to improve industrial remediation by Euglena gracilis (Euglenophyceae), no such roles have been elucidated for this highly adaptive species and its response to heavy metals. This study demonstrates that toxic metals (nickel, lead, cadmium) modify hormonal activity profiles (i.e., CK forms and their concentrations) in E. gracilis. Furthermore, exogenous ABA or CK (tZ) enabled higher metal uptake efficiency (i.e., 9.35% in lead and 9.2% in cadmium uptake with CK) and alleviated metal toxicity through the regulation of endogenous CKs (i.e., total CK, isoprenoid CK) and gibberellin (GAs, GA1 and GA3) levels. These responses suggest that E. gracilis regulates multiple phytohormone signals during metal stress acclimation. A deeper approach, using untargeted metabolomic analyses, gave more detailed insight into phytohormone-controlled pathways and associated modified metabolites, which were frequently related to metal accumulation and the physiological acclimation to metal presence. Significant changes in the levels of cellular metabolites, especially those involved in acclimation to metal stress, were under the influence of phytohormones in algal cells. When grown under metal stress conditions, the presence of exogenous ABA or CKs, caused changes in cellular metabolites which included those from: lipid pathways, riboflavin metabolism, the biosynthesis of cofactors/vitamins, and carbohydrate metabolism. Also, bioactive secondary metabolites (e.g., terpenoids, alkaloids, flavonoids, carotenoids) were modified in algal cells treated with phytohormones. Thus, the study gives a detailed view on the regulatory functions of ABA and CKs in algal metal bioremediation strategies, which are attributed to enhanced metal uptake and in the fine-tuning of plant hormone levels during metal stress response. The results can guide efforts to develop efficient, low-cost and environmentally friendly methods for bioremediation.PMID:36682616 | DOI:10.1016/j.envpol.2023.121094

Am J Obstet Gynecol MFM. 2023 Jan 19:100866. doi: 10.1016/j.ajogmf.2023.100866. Online ahead of print.NO ABSTRACTPMID:36682456 | DOI:10.1016/j.ajogmf.2023.100866

Phytomedicine. 2023 Jan 10;111:154656. doi: 10.1016/j.phymed.2023.154656. Online ahead of print.ABSTRACTBACKGROUD: Bidirectional communications between the gut microbiota and the brain may play a critical role in diabetes-related cognitive impairment. Compound Danshen Dripping Pills (CDDP) treatment has shown remarkable improvement in cognitive impairment in people with type 2 diabetes mellitus (T2DM) in clinical settings, but the underlying mechanisms remain unknown.PURPOSE: An extensive detailed strategy via in vivo functional experiments, transcriptomics, metabolomics, and network pharmacology was adopted to investigate the CDDP-treatment mechanism in diabetic cognitive dysfunction.METHODS: For 12 weeks, KK-Ay mice, a spontaneous T2DM model, were intragastrically administered various doses of CDDP solution or an equivalent volume of water, and the nootropic drug piracetam was orally administered as a positive control. At the 12th week, cognition was assessed using Morris water maze tests and brain magnetic resonance imaging (MRI). Furthermore, transcriptomics, metabolomics, and network pharmacology analyses were applied to reveal novel molecular mechanisms of CDDP-treatment in diabetic cognitive dysfunction of KK-Ay mice, which were then validated using quantitative real-time polymerase chain reaction and Western blot.RESULTS: Here we verified that CDDP can suppress inflammatory response and alleviate the cognitive dysfunction in KK-Ay mice. Also, as demonstrated by 16S rRNA sequencing and short-chain fatty acids (SCFAs) analysis, CDDP attenuated intestinal flora disorder as well as increases of metabolites including butyric acid, hexanoic acid, and isohexic acid. Given the integrated analyses of network pharmacology, transcriptomic, metabolomic data, and molecular biology, the TLR4/MyD88/NF-κB signaling pathway was activated in diabetes, which could be reversed by CDDP.CONCLUSIONS: Our findings demonstrate that CDDP restructures the gut microbiota composition and increased the intestinal SCFAs in KK-Ay mice, which might inhibit neuroinflammation, and thus improve diabetic mice cognitive disorder.PMID:36682300 | DOI:10.1016/j.phymed.2023.154656

J Hazard Mater. 2023 Jan 20;447:130788. doi: 10.1016/j.jhazmat.2023.130788. Online ahead of print.ABSTRACTCombined pollution from microplastics (MPs) and cadmium (Cd) can influence soil environment and soil biota, altering plant growth and development, and Cd mobilization. We investigated the effects of polystyrene (PS) and polypropylene (PP) MPs alongside Cd on soil Cd bioavailability, rhizosphere soil metabolomics, bacterial community structure, and maize (Zea mays L.) growth in two soil types (red soil and cinnamon soil). Although the addition of PS/PP-Cd promoted Cd accumulation in maize plants overall, there were large-particle-size- and small-particle-size-dependent effects in the red soil and cinnamon soil, respectively. The difference is mainly due to the capacity of the large particle size MPs to significantly reduce soil pH, improve soil electrical conductivity (EC), promote active Cd, and intensify Cd mobilization in red soil. In contrast, small-size MPs in cinnamon soil promoted the synthesis and secretion of rhizosphere amino acids and soil metabolites, thus promoting Cd absorption by maize roots. Soil microorganisms also improved Cd bioavailability via C-related functional bacteria. Overall, our study provides novel insights on the potential effects of combined MPs and Cd pollution on soil ecology and agricultural production, enhancing our understanding of rhizosphere metabolites in different soils.PMID:36682251 | DOI:10.1016/j.jhazmat.2023.130788

J Transl Med. 2023 Jan 21;21(1):40. doi: 10.1186/s12967-022-03860-3.ABSTRACTBACKGROUND: Current diagnosis tools for prostate cancer (PCa) such as serum PSA detection and prostate biopsy cannot distinguish dormant tumors from invasive malignancies, either be used as prognosis marker for castration resistant prostate cancer (CRPC), the lethal stage of PCa patients. Exosomes have been widely investigated as promising biomarkers for various diseases. We aim to characterize the proteomic and metabolomic profile of exosomes and to evaluate their potential value for the diagnosis of PCa, especially CRPC. We also investigate the functions of some specific exosome biomarkers in the progression of CRPC.METHODS: Integrated proteomics and metabolomics analysis were performed for plasma-derived exosomes collected from tumor-free controls (TFC), PCa and CRPC patients. Expression of specific exosomal proteins were further validated by targeted 4D-parallel reaction monitoring (PRM) mass spectrometry among the three cohorts. Tissue distribution and functional role of exosomal protein LRG1 was studied in clinical PCa tissue samples and cell line models.RESULTS: Three potential exosomal protein markers were identified. The apolipoprotein E level in PCa samples was 1.7-fold higher than that in TFC (receiver operating characteristic value, 0.74). Similarly, the levels of exosome-derived leucine-rich alpha2-glycoprotein 1 (LRG1) and inter-alpha-trypsin inhibitor heavy chain H3 (ITIH3) in the CRPC group were 1.7 and 2.04 times, respectively, higher than those in the PCa group (ROC values, 0.84 and 0.85, respectively), indicating that LRG1 and ITIH3 could serve as predictive markers for CRPC. For metabolomic evaluation of exosomes, a series of differentially expressed metabolites were identified, and a combined metabolite panel showed ROC value of 0.94 for distinguishing PCa from TFC and 0.97 for distinguishing CRPC from PCa. Immunohistochemistry of tissue microarray showed that LRG1 protein was significantly upregulated in advanced prostate cancer and functional assay revealed that ectopic expression of LRG1 can significantly enhance the malignant phenotype of prostate cancer cells. More importantly, PCa cell derived LRG1-overexpressed exosomes remarkably promoted angiogenesis.CONCLUSION: Integration of proteomics and metabolomics data generated proteomic and metabolic signatures of plasma exosomes that may facilitate discrimination of CRPC from PCa and TFC patients, suggesting the potential of exosomal proteins and metabolites as CRPC markers. The study also confirmed the important role of exosomal protein LRG1 in PCa malignant progression.PMID:36681849 | DOI:10.1186/s12967-022-03860-3

Signal Transduct Target Ther. 2023 Jan 21;8(1):41. doi: 10.1038/s41392-023-01311-z.ABSTRACTUrinary stone is conceptualized as a chronic metabolic disorder punctuated by symptomatic stone events. It has been shown that the occurrence of calcium oxalate monohydrate (COM) during stone formation is regulated by crystal growth modifiers. Although crystallization inhibitors have been recognized as a therapeutic modality for decades, limited progress has been made in the discovery of effective modifiers to intervene with stone disease. In this study, we have used metabolomics technologies, a powerful approach to identify biomarkers by screening the urine components of the dynamic progression in a bladder stone model. By in-depth mining and analysis of metabolomics data, we have screened five differential metabolites. Through density functional theory studies and bulk crystallization, we found that three of them (salicyluric, gentisic acid and succinate) could effectively inhibit nucleation in vitro. We thereby assessed the impact of the inhibitors with an EG-induced rat model for kidney stones. Notably, succinate, a key player in the tricarboxylic acid cycle, could decrease kidney calcium deposition and injury in the model. Transcriptomic analysis further showed that the protective effect of succinate was mainly through anti-inflammation, inhibition of cell adhesion and osteogenic differentiation. These findings indicated that succinate may provide a new therapeutic option for urinary stones.PMID:36681678 | DOI:10.1038/s41392-023-01311-z

Food Microbiol. 2023 May;111:104193. doi: 10.1016/j.fm.2022.104193. Epub 2022 Nov 25.ABSTRACTAlthough the impact of nitrogen nutrition on the production of fermentative aromas in oenological fermentation is well known today, one may wonder whether the effects studied are the same when winemaking takes place at high turbidities, specifically for the production of wines intended for cognac distillation. To that effect, a fermentation robot was used to analyze 30 different fermentation conditions at two turbidity levels with several factors tested: (i) initial addition of nitrogen either organic (with a mixture of amino acids - MixAA) or inorganic with di-ammonium phosphate (DAP) at different concentrations, (ii) variation of the ratio of inorganic/organic nitrogen (MixAA and DAP) and (iii) addition of different single amino acids (alanine, arginine, aspartic acid and glutamic acid). A metabolomic analysis was carried out on all resulting wines to have a global vision of the impact of nitrogen on more than sixty aromatic molecules of various families. Then, at the end of the alcoholic fermentation, the wines were micro-distilled. A first interesting observation was that the aroma profiles of both wines and distillates were close, indicating that the concentration factor is rather similar for the different aromas studied. Secondly, the fermentation kinetics and aroma results have shown that the nitrogen concentration effect prevailed over the nature of nitrogen. Although the lipid concentration was in excess, an interaction between the assimilable nitrogen and lipid contents was still observed in wines or in micro-distillates. Alanine is involved in the synthesis of acetaldehyde, isobutanol, isoamyl alcohol and isoamyl acetate. Finally, it was demonstrated that modifying the ratio of assimilable nitrogen in musts is not an interesting technological response to improve the aromatic profile of wines and brandies. Indeed, unbalance the physiological ratio of the must by adding a single source of assimilable nitrogen (organic or inorganic) has been shown to deregulate the synthesis of most of the fermentation aromas produced by the yeast. Wine metabolomic analysis confirmed the results that had been observed in micro-distillates but also in the other aromatic families, especially on terpenes. The contribution of solid particles, but also yeast biosynthesis (via sterol management in must) to wine terpenes is discussed. Indeed, the synthesis of terpenes in this oenological context seems to be favored, especially since the concentration of assimilable nitrogen (in addition to the lipid content) favor their accumulation in the medium. A non-negligible vintage effect on the terpene profile was also demonstrated with variations in their distribution depending on the years. Thus, the present study focuses on the metabolism of wine yeasts under different environmental conditions (nitrogen and lipid content) and on the impact of distillation on the fate of flavor compounds. The results highlight once again the complexity of metabolic fluxes and of the impact of nitrogen source (nature and amount) and of lipids. Furthermore, this study demonstrates that beyond the varietal origin of terpenes, the part resulting from the de novo synthesis by the yeast during the fermentation cannot be neglected in the context of cognac winemaking with high levels of turbidity.PMID:36681397 | DOI:10.1016/j.fm.2022.104193

Food Chem Toxicol. 2023 Jan 18:113612. doi: 10.1016/j.fct.2023.113612. Online ahead of print.ABSTRACTThe toxicity effects of chiral pesticides on living organisms have attracted an increasing public attention. This study aims to investigate the toxicity effect and mechanism of hexaconazole (HEX) to human breast cancer cell (MCF-7) at enantiomer levels. HEX exposure obviously inhibited cells activities in a dose-dependent manner. Under the conditions of VIP >1 and p < 0.05, a total of 255 and 177 differential metabolites (DMs), 17 and 15 amino acid- and lipid-related metabolic pathways were disturbed after (+)-HEX and (-)-HEX exposure, respectively. HEX exposure may affect cell membrane function, signal transduction, and cell differentiation. We further investigated the mechanism of enantioselective differences by using molecular docking which showed that CYP17A1 was the main enzyme that leading to endocrine disrupting effects with the binding energy of -6.30 and -6.08 kcal/mol compared to CYP19A1 enzyme which were -5.81 and -5.93 kcal/mol for (+)-HEX and (-)-HEX, respectively. The docking results explained the reasons why (+)-HEX achieved higher cytotoxicity and induced more seriously metabolic profiles than its antipode. These findings could provide a new insight to understand the enantioselective cytotoxicity effect and mechanism of HEX and will be conducive to assessing its risk to human health at enantiomer levels.PMID:36681264 | DOI:10.1016/j.fct.2023.113612

Plant Physiol Biochem. 2023 Jan 17;195:351-361. doi: 10.1016/j.plaphy.2023.01.028. Online ahead of print.ABSTRACTRadish is an important taproot crop with medicinal and edible uses that is cultivated worldwide. However, the differences in metabolites and the underlying molecular bases among different radish types remain largely unknown. In the present study, a combined analysis of liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) and RNA-Seq data was conducted to uncover important differentially accumulated metabolites (DAMs) among radish accessions with green, white and red taproot flesh colours. A total of 657 metabolites were identified and 138 DAMs were commonly present in red vs. green and red vs. white accessions. Red accessions were rich in phenolic compounds, while green and white accessions had more amino acids. Additionally, 41 metabolites and 98 genes encoding 37 enzymes were enriched in the shikimate and phenolic biosynthesis pathways. CHS is the rate-limiting enzyme determining flavonoid differences among accessions. A total of 119 candidate genes might contribute to red accession-specific accumulated metabolites. Specifically, one gene cluster consisting of 16 genes, including one RsMYB1, RsMYBL2, RsTT8, RsDFR, RsANS, Rs4CL3, RsSCPL10, Rs3AT1 and RsSAP2 gene, two RsTT19 and RsWRKY44 genes and three RsUGT genes, might be involved in anthocyanin accumulation in red radish fleshy taproots. More importantly, an InDel marker was developed based on an RsMYB1 promoter mutation, and the accuracy reached 95.9% when it was used to select red-fleshed radishes. This study provides comprehensive insights into the metabolite differences and underlying molecular mechanisms in fleshy taproots among different radish genotypes and will be beneficial for the genetic improvement of radish nutritional quality.PMID:36681065 | DOI:10.1016/j.plaphy.2023.01.028

J Chromatogr A. 2023 Jan 11;1690:463779. doi: 10.1016/j.chroma.2023.463779. Online ahead of print.ABSTRACTUntargeted metabolomic studies require an extensive set of analyte (metabolic) information to be obtained from each analyzed sample. Thus, highly selective, and efficient analytical methodologies together with reversed-phase (RP) or hydrophilic interaction liquid chromatography (HILIC) are usually applied in these approaches. Here, we present a performance comparison of five different chromatographic columns (C18, C8, RP Amide, zicHILIC, OH5 HILIC phases) to evaluate their sufficiency of analysis for a large analyte library, consisting of 817 authentic standards. By taking into account experimental chromatographic parameters (i.e. retention time, peak tailing and asymmetry, FWHM, signal-to-noise ratio and peak area and intensity), the proposed column scoring approach provides a simple criterion that may assist analysis in the select of a stationary phase for those metabolites of interest. RPLC methods offered better results regarding metabolic library coverage, while the zicHILIC stationary phase delivered a bigger number of properly eluted compounds. This study demonstrates the importance of choosing the most suitable configuration for the analysis of different metabolic classes.PMID:36681007 | DOI:10.1016/j.chroma.2023.463779

Planta. 2023 Jan 21;257(2):41. doi: 10.1007/s00425-022-04065-0.ABSTRACTSevere N stress allows an accumulation of C-based compounds but impedes that of N-based compounds required to lower the susceptibility of tomato stem to Botrytis cinerea. Botrytis cinerea, a necrotrophic filamentous fungus, forms potentially lethal lesions on the stems of infected plants. Contrasted levels of susceptibility to B. cinerea were obtained in a tomato cultivar grown on a range of nitrate concentration: low N supply resulted in high susceptibility while high N supply conferred a strong resistance. Metabolic deviations and physiological traits resulting from both infection and nitrogen limitation were investigated in the symptomless stem tissue surrounding the necrotic lesion. Prior to infection, nitrogen-deficient plants showed reduced levels of nitrogen-based compounds such as amino acids, proteins, and glutathione and elevated levels of carbon-based and defence compounds such as α-tomatine and chlorogenic acid. After B. cinerea inoculation, all plants displayed a few common responses, mainly alanine accumulation and galactinol depletion. The metabolome of resistant plants grown under high N supply showed no significant change after inoculation. On the contrary, the metabolome of susceptible plants grown under low N supply showed massive metabolic adjustments, including changes in central metabolism around glutamate and respiratory pathways, suggesting active resource mobilization and production of energy and reducing power. Redox and defence metabolisms were also stimulated by the infection in plants grown under low N supply; glutathione and chlorogenic acid accumulated, as well as metabolites with more controversial defensive roles, such as polyamines, GABA, branched-chain amino acids and phytosterols. Taken together, the results showed that nitrogen deficiency, although leading to an increase in secondary metabolites even before the pathogen attack, must have compromised the constitutive levels of defence proteins and delayed or attenuated the induced responses. The involvement of galactinol, alanine, cycloartenol and citramalate in the tomato stem response to B. cinerea is reported here for the first time.PMID:36680621 | DOI:10.1007/s00425-022-04065-0

J Inherit Metab Dis. 2023 Jan 21. doi: 10.1002/jimd.12591. Online ahead of print.ABSTRACTImproved second-tier assays are needed to reduce the number of false positives in newborn screening (NBS) for inherited metabolic disorders including those on the Recommended Uniform Screening Panel (RUSP). We developed an expanded metabolite panel for second-tier testing of dried blood spot (DBS) samples from screen-positive cases reported by the California NBS program, consisting of true- and false-positives from four disorders: glutaric acidemia type 1 (GA1), methylmalonic acidemia (MMA), ornithine transcarbamylase deficiency (OTCD), and very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). This panel was assembled from known disease markers and new features discovered by untargeted metabolomics and applied to second-tier analysis of single DBS punches using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a 3-min run. Additionally, we trained a Random Forest (RF) machine learning classifier to improve separation of true- and false positive cases. Targeted metabolomic analysis of 121 analytes from DBS extracts in combination with RF classification at a sensitivity of 100% reduced false positives for GA1 by 83%, MMA by 84%, OTCD by 100%, and VLCADD by 51%. This performance was driven by a combination of known disease markers (3-hydroxyglutaric acid, methylmalonic acid, citrulline, C14:1), other amino acids and acylcarnitines, and novel metabolites identified to be isobaric to several long-chain acylcarnitine and hydroxy-acylcarnitine species. These findings establish the effectiveness of this second-tier test to improve screening for these four conditions and demonstrate the utility of supervised machine learning in reducing false-positives for conditions lacking clearly discriminating markers, with future studies aimed at optimizing and expanding the panel to additional disease targets. This article is protected by copyright. All rights reserved.PMID:36680545 | DOI:10.1002/jimd.12591

Viruses. 2023 Jan 14;15(1):242. doi: 10.3390/v15010242.ABSTRACTInfluenza represents a major and ongoing public health hazard. Current collaborative efforts are aimed toward creating a universal flu vaccine with the goals of both improving responses to vaccination and increasing the breadth of protection against multiple strains and clades from a single vaccine. As an intermediate step toward these goals, the current work is focused on evaluating the systemic host response to vaccination in both normal and high-risk populations, such as the obese and geriatric populations, which have been linked to poor responses to vaccination. We therefore employed a metabolomics approach using a time-course (n = 5 time points) of the response to human vaccination against influenza from the time before vaccination (pre) to 90 days following vaccination. We analyzed the urinary profiles of a cohort of subjects (n = 179) designed to evenly sample across age, sex, BMI, and other demographic factors, stratifying their responses to vaccination as "High", "Low", or "None" based on the seroconversion measured by hemagglutination inhibition assay (HAI) from plasma samples at day 28 post-vaccination. Overall, we putatively identified 15,903 distinct, named, small-molecule structures (4473 at 10% FDR) among the 895 samples analyzed, with the aim of identifying metabolite correlates of the vaccine response, as well as prognostic and diagnostic markers from the periods before and after vaccination, respectively. Notably, we found that the metabolic profiles could unbiasedly separate the high-risk High-responders from the high-risk None-responders (obese/geriatric) within 3 days post-vaccination. The purine metabolites Guanine and Hypoxanthine were negatively associated with high seroconversion (p = 0.0032, p &lt; 0.0001, respectively), while Acetyl-Leucine and 5-Aminovaleric acid were positively associated. Further changes in Cystine, Glutamic acid, Kynurenine and other metabolites implicated early oxidative stress (3 days) after vaccination as a hallmark of the High-responders. Ongoing efforts are aimed toward validating these putative markers using a ferret model of influenza infection, as well as an independent cohort of human seasonal vaccination and human challenge studies with live virus.PMID:36680282 | DOI:10.3390/v15010242

Viruses. 2022 Dec 30;15(1):121. doi: 10.3390/v15010121.ABSTRACTMixed virus infections threaten crop production because interactions between the host and the pathogen mix may lead to viral synergism. While individual infections by potato virus A (PVA), a potyvirus, and potato virus X (PVX), a potexvirus, can be mild, co-infection leads to synergistic enhancement of PVX and severe symptoms. We combined image-based phenotyping with metabolite analysis of single and mixed PVA and PVX infections and compared their effects on growth, photosynthesis, and metabolites in Nicotiana benthamiana. Viral synergism was evident in symptom severity and impaired growth in the plants. Indicative of stress, the co-infection increased leaf temperature and decreased photosynthetic parameters. In contrast, singly infected plants sustained photosynthetic activity. The host's metabolic response differed significantly between single and mixed infections. Over 200 metabolites were differentially regulated in the mixed infection: especially defense-related metabolites and aromatic and branched-chain amino acids increased compared to the control. Changes in the levels of methionine cycle intermediates and a low S-adenosylmethionine/S-adenosylhomocysteine ratio suggested a decline in the methylation potential in co-infected plants. The decreased ratio between reduced glutathione, an important scavenger of reactive oxygen species, and its oxidized form, indicated that severe oxidative stress developed during co-infection. Based on the results, infection-associated oxidative stress is successfully controlled in the single infections but not in the synergistic infection, where activated defense pathways are not sufficient to counter the impact of the infections on plant growth.PMID:36680161 | DOI:10.3390/v15010121

Plants (Basel). 2023 Jan 15;12(2):408. doi: 10.3390/plants12020408.ABSTRACTSystems biology has been increasingly applied with multiple omics for a holistic comprehension of complex biological systems beyond the reductionist approach that focuses on individual molecules. Different high-throughput omics approaches, including genomics, transcriptomics, metagenomics, proteomics, and metabolomics have been implemented to study the molecular mechanisms of botanical carnivory. This covers almost all orders of carnivorous plants, namely Caryophyllales, Ericales, Lamiales, and Oxalidales, except Poales. Studies using single-omics or integrated multi-omics elucidate the compositional changes in nucleic acids, proteins, and metabolites. The omics studies on carnivorous plants have led to insights into the carnivory origin and evolution, such as prey capture and digestion as well as the physiological adaptations of trap organ formation. Our understandings of botanical carnivory are further enhanced by the discoveries of digestive enzymes and transporter proteins that aid in efficient nutrient sequestration alongside dynamic molecular responses to prey. Metagenomics studies revealed the mutualistic relationships between microbes and carnivorous plants. Lastly, in silico analysis accelerated the functional characterization of new molecules from carnivorous plants. These studies have provided invaluable molecular data for systems understanding of carnivorous plants. More studies are needed to cover the diverse species with convergent evolution of botanical carnivory.PMID:36679121 | DOI:10.3390/plants12020408

Plants (Basel). 2023 Jan 15;12(2):404. doi: 10.3390/plants12020404.ABSTRACTThe Plasmopara viticola pathogen causes one of the most severe grapevine diseases, namely downy mildew. The response to P. viticola involves both visible symptoms and intricate metabolomic alterations, particularly in relation to volatile organic compounds, and depends on the degree of resistance of a particular variety. There are numerous native grapevine varieties in Croatia, and they vary in susceptibility to this oomycete. As previously reported, in vitro leaf disc bioassay and polyphenolic compound analysis are complementary methods that can be used to separate native varieties into various resistance classes. This research used the Solid Phase Microextraction-Arrow Gas Chromatography-Mass Spectrometry method to identify the early alterations in the VOCs in the leaves after P. viticola inoculation. Based on the absolute peak area of sesquiterpenes, some discrepancies between the sampling terms were noticed. The presence of certain chemical compounds such as humulene, ylangene, and α-farnesene helped distinguish the non-inoculated and inoculated samples. Although specific VOC responses to P. viticola infection of native varieties from various resistance classes could not be identified, the response of less susceptible native varieties and resistant controls was associated with an increase in the absolute peak area of several compounds, including geranylacetone, ß-ocimene, and (E)-2-hexen-1-ol.PMID:36679116 | DOI:10.3390/plants12020404

Plants (Basel). 2023 Jan 10;12(2):325. doi: 10.3390/plants12020325.ABSTRACTBrassica napus L. is a vital plant oil resource worldwide. The fatty acid biosynthesis and oil accumulation in its seeds are controlled by several genetic and environmental factors, including daytime and nighttime temperatures. We analyzed changes in oleic and erucic acid content in two double haploid (DH) lines, DH0729, a weakly temperature-sensitive line, and DH0815, a strongly temperature-sensitive line, derived from B. napus plants grown at different altitudes (1600, 1800, 2000, 2200, and 2400 m a.s.l., 28.85° N, 112.35° E) and nighttime temperatures (20/18, 20/16, 20/13 and 20/10 °C, daytime/nighttime temperature). Based on medium- and long-chain fatty acid metabolites, the total oleic acid content 35 and 43 days after flowering was significantly lower in low nighttime temperature (LNT, 20/13 °C) plants than in high nighttime temperature (HNT, 20/18 °C) plants (HNT: 58-62%; LNT: 49-54%; an average decrease of 9%), and the total erucic acid content was significantly lower in HNT than in LNT plants (HNT: 1-2%; LNT: 8-13%; an average increase of 10%). An RNA-seq analysis showed that the expression levels of SAD (LOC106366808), ECR (LOC106396280), KCS (LOC106419344), KAR (LOC106367337), HB1(LOC106430193), and DOF5 (LOC111211868) in STSL seeds increased under LNT conditions. In STSL seeds, a base mutation in the cis-acting element involved in low-temperature responsiveness (LTR), the HB1 and KCS promoter caused loss of sensitivity to low temperatures, whereas that of the KCS promoter caused increased sensitivity to low temperatures.PMID:36679038 | DOI:10.3390/plants12020325