Molecular analysis of broad-spectrum induced resistance in rice by the green leaf volatile Z-3-hexenyl acetate.

Green leaf volatiles (GLVs), volatile organic compounds released by plants upon tissue damage, are key signaling molecules in plant immunity. The ability of exogenous GLV application to trigger an induced resistance (IR) phenotype against arthropod pests has been widely reported, but its effectiveness against plant pathogens is less well understood. In this study, we combined mRNA sequencing-based transcriptomics and phytohormone measurements with multispectral imaging-based precision phenotyping to gain insights into the molecular basis of Z-3-hexenyl acetate-induced resistance (Z-3-HAC-IR) in rice. Furthermore, we evaluated the efficacy of Z-3-HAC-IR against a panel of economically significant rice pathogens: Pyricularia oryzae, Rhizoctonia solani, Xanthomonas oryzae pv. oryzae, Cochliobolus miyabeanus, and Meloidogyne graminicola. Our data revealed rapid induction of jasmonate metabolism and systemic induction of plant immune responses upon Z-3-HAC exposure, as well as a transient allocation cost due to accelerated chlorophyll degradation and nutrient remobilization. Z-3-HAC-IR proved effective against all tested pathogens except for C. miyabeanus, including against the (hemi)biotrophs M. graminicola, X. oryzae pv. oryzae, and P. oryzae. The Z-3-HAC-IR phenotype was lost in the jasmonate (JA)-deficient hebiba mutant, which confirms the causal role of JA in Z-3-HAC-IR. Together, our results show that GLV exposure in rice induces broad-spectrum, JA-mediated disease resistance with limited allocation costs, and may thus be a promising alternative crop protection approach.

Paediatric obesity: a systematic review and pathway mapping of metabolic alterations underlying early disease processes.

BACKGROUND: The alarming trend of paediatric obesity deserves our greatest awareness to hinder the early onset of metabolic complications impacting growth and functionality. Presently, insight into molecular mechanisms of childhood obesity and associated metabolic comorbidities is limited. This systematic review aimed at scrutinising what has been reported on putative metabolites distinctive for metabolic abnormalities manifesting at young age by searching three literature databases (Web of Science, Pubmed and EMBASE) during the last 6 years (January 2015-January 2021). Global metabolomic profiling of paediatric obesity was performed (multiple biological matrices: blood, urine, saliva and adipose tissue) to enable overarching pathway analysis and network mapping. Among 2792 screened Q1 articles, 40 met the eligibility criteria and were included to build a database on metabolite markers involved in the spectrum of childhood obesity. Differential alterations in multiple pathways linked to lipid, carbohydrate and amino acid metabolisms were observed. High levels of lactate, pyruvate, alanine and acetate marked a pronounced shift towards hypoxic conditions in children with obesity, and, together with distinct alterations in lipid metabolism, pointed towards dysbiosis and immunometabolism occurring early in life. Additionally, aberrant levels of several amino acids, most notably belonging to tryptophan metabolism including the kynurenine pathway and its relation to histidine, phenylalanine and purine metabolism were displayed. Moreover, branched-chain amino acids were linked to lipid, carbohydrate, amino acid and microbial metabolism, inferring a key role in obesity-associated insulin resistance. CONCLUSIONS: This systematic review revealed that the main metabolites at the crossroad of dysregulated metabolic pathways underlying childhood obesity could be tracked down to one central disturbance, i.e. impending insulin resistance for which reference values and standardised measures still are lacking. In essence, glycolytic metabolism was evinced as driving energy source, coupled to impaired Krebs cycle flux and ß-oxidation. Applying metabolomics enabled to retrieve distinct metabolite alterations in childhood obesity(-related insulin resistance) and associated pathways at early age and thus could provide a timely indication of risk by elucidating early-stage biomarkers as hallmarks of future metabolically unhealthy phenotypes.

Validated Ultra-High-Performance Liquid Chromatography Hybrid High-Resolution Mass Spectrometry and Laser-Assisted Rapid Evaporative Ionization Mass Spectrometry for Salivary Metabolomics.

Whereas urine and blood are typically targeted in clinical research, saliva represents an interesting alternative because its intrinsic metabolome is chemically diverse and reflective for various biological processes. Moreover, saliva collection is easy and noninvasive, which is especially valuable for cohorts in which sample collection is challenging, for example, infants and children. With this rationale, we established a validated ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) method for salivary metabolic profiling and fingerprinting. Hereby, 450 µL of saliva was centrifuged and passed over a 0.45-µm polyamide membrane filter, after which the extract was subjected to chromatographic analysis (HSS T3 column) and Q-Exactive Orbitrap-MS. For the majority of the profiled metabolites, good linearity (R2 ≥ 0.99) and precision (coefficient of variance ≤ 15%) was achieved. The fingerprinting performance was evaluated based on the complete metabolome (11 385 components), whereby 76.8% was found compliant with the criteria for precision (coefficient of variance ≤ 30%) and 82.7% with linearity (R2 ≥ 0.99). In addition, the method was proven fit-for-purpose for a cohort of 140 adolescents (6-16 years, stratified according to weight), yielding relevant profiles (45 obesity-related metabolites) and discriminative fingerprints (Q2 of 0.784 for supervised discriminant analysis). Alternatively, laser-assisted rapid evaporative ionization mass spectrometry (LA-REIMS) was established for rapid fingerprinting of saliva, thereby using a Nd:YAG laser and Xevo G2-XS QToF-MS. With an acquisition time of 0.5 min per sample, LA-REIMS offers unique opportunities for point-of-care applications. In conclusion, this work presents a platform of UHPLC-HRMS and LA-REIMS, complementing each other to perform salivary metabolomics.

Gibberellin antagonizes jasmonate-induced defense against Meloidogyne graminicola in rice.

Gibberellin (GA) regulates various plant growth and developmental processes, but its role in pathogen attack, and especially nematode-plant interactions, still remains to be elucidated. An in-depth characterization of the role of GA in nematode infection was conducted using mutant lines of rice, chemical inhibitors, and phytohormone measurements. Our results showed that GA influences rice-Meloidogyne graminicola interactions in a concentration-dependent manner. Foliar spray of plants with a low concentration of gibberellic acid enhanced nematode infection. Biosynthetic and signaling mutants confirmed the importance of gibberellin for rice susceptibility to M. graminicola infection. Our study also demonstrates that GA signaling suppresses jasmonate (JA)-mediated defense against M. graminicola, and likewise the JA-induced defense against M. graminicola requires SLENDER RICE1 (SLR1)-mediated repression of the GA pathway. In contrast to observations from other plant-pathogen interactions, GA plays a dominant role over JA in determining susceptibility to M. graminicola in rice. This GA-induced nematode susceptibility was largely independent of auxin biosynthesis, but relied on auxin transport. In conclusion, we showed that GA-JA antagonistic crosstalk is at the forefront of the interaction between rice and M. graminicola, and SLR1 plays a central role in the JA-mediated defense response in rice against this nematode.

Holistic Lipidomics of the Human Gut Phenotype Using Validated Ultra-High-Performance Liquid Chromatography Coupled to Hybrid Orbitrap Mass Spectrometry.

As lipids are assigned a plethora of biological functions, it is evident that dysregulated lipid metabolism signifies a key element in many pathological conditions. With this rationale, this study presents a validated lipidomics platform to map the fecal lipidome, which integrates unique information about host-gut microbiome interactions, gastrointestinal functionality, and dietary patterns. This particular method accomplished coverage across all eight lipid categories: fatty acyls, glycerolipids, phosphoglycerolipids, polyketides, prenols, saccharolipids, sphingolipids, and sterols. Generic extraction of freeze-dried feces was achieved by solid-liquid extraction using methanol and methyl tert-butyl ether. Extracted components were separated by liquid chromatography, whereby the selected ethylene-bridged hybrid phenyl ultra-high-performance liquid chromatography stationary phase allowed fast separation of both individual lipid species and categories. Detection was achieved by high-resolution full-scan Q-Exactive Orbitrap mass spectrometry and covered a broad m/z scan range (67-2300 Da). Method validation was performed in a targeted fashion to evaluate the analytical performance across all lipid categories, revealing excellent linearity (R2 ≥ 0.9921), acceptable repeatability (coefficients of variance ≤15.6%), and stable recovery (coefficients of variance ≤11.9%). Method suitability for untargeted fingerprinting was verified, demonstrating adequate linearity (R2 ≥ 0.90) for 75.3% and acceptable repeatability (coefficients of variance ≤30%) for 84.5% of about 9000 endogenous fecal compounds. Eventually, the potential of fecal lipidomics was exemplified within a clinical context of type 2 diabetes, thereby revealing significant perturbations [orthogonal partial least-squares discriminant analysis Q2(Y) of 0.728] in the fecal lipidome between participants with normal blood glucose levels (n = 26) and those with type 2 diabetes (n = 17).

Pharmacokinetic and urinary profiling reveals the prednisolone/cortisol ratio as a valid biomarker for prednisolone administration.

BACKGROUND: In Europe, synthetic corticosteroids are not allowed in animal breeding for growth-promoting purposes. Nevertheless, a high prevalence of non-compliant urine samples was recently reported for prednisolone, however, without any indication of unauthorized use. Within this context, 20ß-dihydroprednisolone and the prednisolone/cortisol ratio have been suggested as potential tools to discriminate between exogenous and endogenous urinary prednisolone. In this study, the validity of these strategies was verified by investigating the plasma pharmacokinetic and urinary excretion profiles of relevant glucocorticoids in bovines, subjected to exogenous prednisolone treatment or tetracosactide hexaacetate administration to induce endogenous prednisolone formation. Bovine urine and plasma samples were analysed by liquid chromatography and mass spectrometry. RESULTS: Based on the plasma pharmacokinetics and urinary profiles, 20ß-dihydroprednisolone was confirmed as the main prednisolone-derived metabolite, being detected in the biological fluids of all 12 bovines (plasma AUC0-inf of 121 h µg L-1 and urinary concentration > 0.695 µg L-1). However, this metabolite enclosed no potential as discriminative marker as no significant concentration differences were observed upon exogenous prednisolone treatment or tetracosactide hexaacetate administration under all experimental conditions. As a second marker tool, the prednisolone/cortisol ratios were assessed along the various treatments, taking into account that endogenous prednisolone formation involves the hypothalamic-pituitary-adrenal axis and is associated with an increased cortisol secretion. Significantly lower ratios were observed in case of endogenous prednisolone formation (i.e. ratios ranging from 0.00379 to 0.129) compared to the exogenous prednisolone treatment (i.e. ratios ranging from 0.0603 to 36.9). On the basis of these findings, a discriminative threshold of 0.260 was proposed, which allowed classification of urine samples according to prednisolone origin with a sensitivity of 94.2% and specificity of 99.0%. CONCLUSION: The prednisolone/cortisol ratio was affirmed as an expedient strategy to discriminate between endogenous and exogenous prednisolone in urine. Although the suggested threshold value was associated with high specificity and sensitivity, a large-scale study with varying experimental conditions is designated to optimize this value.

Priming of wheat with the green leaf volatile Z-3-hexenyl acetate enhances defense against Fusarium graminearum but boosts deoxynivalenol production.

Priming refers to a mechanism whereby plants are sensitized to respond faster and/or more strongly to future pathogen attack. Here, we demonstrate that preexposure to the green leaf volatile Z-3-hexenyl acetate (Z-3-HAC) primed wheat (Triticum aestivum) for enhanced defense against subsequent infection with the hemibiotrophic fungus Fusarium graminearum. Bioassays showed that, after priming with Z-3-HAC, wheat ears accumulated up to 40% fewer necrotic spikelets. Furthermore, leaves of seedlings showed significantly smaller necrotic lesions compared with nonprimed plants, coinciding with strongly reduced fungal growth in planta. Additionally, we found that F. graminearum produced more deoxynivalenol, a mycotoxin, in the primed treatment. Expression analysis of salicylic acid (SA) and jasmonic acid (JA) biosynthesis genes and exogenous methyl salicylate and methyl jasmonate applications showed that plant defense against F. graminearum is sequentially regulated by SA and JA during the early and later stages of infection, respectively. Interestingly, analysis of the effect of Z-3-HAC pretreatment on SA- and JA-responsive gene expression in hormone-treated and pathogen-inoculated seedlings revealed that Z-3-HAC boosts JA-dependent defenses during the necrotrophic infection stage of F. graminearum but suppresses SA-regulated defense during its biotrophic phase. Together, these findings highlight the importance of temporally separated hormone changes in molding plant health and disease and support a scenario whereby the green leaf volatile Z-3-HAC protects wheat against Fusarium head blight by priming for enhanced JA-dependent defenses during the necrotrophic stages of infection.

Metabolic Fingerprinting to Assess the Impact of Salinity on Carotenoid Content in Developing Tomato Fruits.

As the presence of health-promoting substances has become a significant aspect of tomato fruit appreciation, this study investigated nutrient solution salinity as a tool to enhance carotenoid accumulation in cherry tomato fruit (Solanum lycopersicum L. cv. Juanita). Hereby, a key objective was to uncover the underlying mechanisms of carotenoid metabolism, moving away from typical black box research strategies. To this end, a greenhouse experiment with five salinity treatments (ranging from 2.0 to 5.0 decisiemens (dS) m(-1)) was carried out and a metabolomic fingerprinting approach was applied to obtain valuable insights on the complicated interactions between salinity treatments, environmental conditions, and the plant's genetic background. Hereby, several hundreds of metabolites were attributed a role in the plant's salinity response (at the fruit level), whereby the overall impact turned out to be highly depending on the developmental stage. In addition, 46 of these metabolites embraced a dual significance as they were ascribed a prominent role in carotenoid metabolism as well. Based on the specific mediating actions of the retained metabolites, it could be determined that altered salinity had only marginal potential to enhance carotenoid accumulation in the concerned tomato fruit cultivar. This study invigorates the usefulness of metabolomics in modern agriculture, for instance in modeling tomato fruit quality. Moreover, the metabolome changes that were caused by the different salinity levels may enclose valuable information towards other salinity-related plant processes as well.

Quantification and profiling of lipophilic marine toxins in microalgae by UHPLC coupled to high-resolution orbitrap mass spectrometry.

During the last decade, a significant increase in the occurrence of harmful algal blooms (HABs), linked to repetitive cases of shellfish contamination has become a public health concern and therefore, accurate methods to detect marine toxins in different matrices are required. In this study, we developed a method for profiling lipophilic marine microalgal toxins based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS). Extraction of selected toxins (okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX) and 13-desmethyl spirolide C (SPX-1)) was optimized using a Plackett-Burman design. Three key algal species, i.e., Prorocentrum lima, Protoceratium reticulatum and Alexandrium ostenfeldii were used to test the extraction efficiency of OA, YTXs and SPXs, respectively. Prorocentrum micans, fortified with certified reference solutions, was used for recovery studies. The quantitative and confirmatory performance of the method was evaluated according to CD 2002/657/EC. Limits of detection and quantification ranged between 0.006 and 0.050 ng mL(-1) and 0.018 to 0.227 ng mL(-1), respectively. The intra-laboratory reproducibility ranged from 6.8 to 11.7 %, repeatability from 6.41 to 11.5 % and mean corrected recoveries from 81.9 to 119.6 %. In addition, algae cultures were retrospectively screened for analogues and metabolites through a homemade database. Using the ToxID software programme, 18 toxin derivates were detected in the extract of three toxin producing microalgae species. In conclusion, the generic extraction and full-scan HRMS approach offers an excellent quantitative performance and simultaneously allows to profile analogues and metabolites of marine toxins in microalgae. Graphical Abstract Optimization of extraction, detection and quantification of lipophilic marine toxins in microalgae by UHPLC-HR Orbitrap MS.

Stem girdling affects the quantity of CO2 transported in xylem as well as CO2 efflux from soil.

There is recent clear evidence that an important fraction of root-respired CO2 is transported upward in the transpiration stream in tree stems rather than fluxing to the soil. In this study, we aimed to quantify the contribution of root-respired CO2 to both soil CO2 efflux and xylem CO2 transport by manipulating the autotrophic component of belowground respiration. We compared soil CO2 efflux and the flux of root-respired CO2 transported in the transpiration stream in girdled and nongirdled 9-yr-old oak trees (Quercus robur) to assess the impact of a change in the autotrophic component of belowground respiration on both CO2 fluxes. Stem girdling decreased xylem CO2 concentration, indicating that belowground respiration contributes to the aboveground transport of internal CO2 . Girdling also decreased soil CO2 efflux. These results confirmed that root respiration contributes to xylem CO2 transport and that failure to account for this flux results in inaccurate estimates of belowground respiration when efflux-based methods are used. This research adds to the growing body of evidence that efflux-based measurements of belowground respiration underestimate autotrophic contributions.

High-resolution Orbitrap mass spectrometry for the analysis of carotenoids in tomato fruit: validation and comparative evaluation towards UV-VIS and tandem mass spectrometry.

In this study, a generic extraction protocol and full-scan high-resolution Orbitrap-mass spectrometry (MS) detection method were developed, enabling the metabolomic screening for carotenoids in tomato fruit tissue. To this end, the carotenoids lutein, zeaxanthin, α-carotene, ß-carotene, and lycopene (representing both xanthofylls and carotenes) were considered. The extraction procedure was optimized by means of a D-optimal design and consisted of a liquid-liquid extraction with methanol/tert-butyl methyl ether (1:1, v/v). The considered compounds were detected by a single-stage Exactive(TM) mass spectrometer, operating at a mass resolution of 100,000 full width at half maximum. The validation study demonstrated excellent performance in terms of linearity (R (2) > 0.99), repeatability (CV ≤ 10.6 %), within-laboratory reproducibility (CV ≤ 12.2 %), and mean corrected recovery (ranging from 85 to 106 %). Additionally, a comparative evaluation towards well-established detection techniques, i.e., tandem mass spectrometry (MS/MS) and ultraviolet-visible spectroscopy (UV-VIS) photodiode array, indicated superior performance of high-resolution Orbitrap-MS with regard to specificity/selectivity and sensitivity (with limits of detection ranging from 1.0 to 3.8 pg µL(-1)). As a result, it may be concluded that high-resolution Orbitrap-MS is a suited alternative for UV-VIS or MS/MS in analyzing carotenoids and may offer significant value in carotenoid research because of the metabolomic screening possibilities.

Validation of a confirmatory method for lipophilic marine toxins in shellfish using UHPLC-HR-Orbitrap MS.

Lipophilic marine toxins are produced by harmful microalgae and can accumulate in edible filter feeders such as shellfish, leading to an introduction of toxins into the human food chain, causing different poisoning effects. During the last years, analytical methods, based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been consolidated by interlaboratory validations. However, the main drawback of LC-MS/MS methods remains the limited number of compounds that can be analyzed in a single run. Due to the targeted nature of these methods, only known toxins, previously considered during method optimization, will be detected. Therefore in this study, a method based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS) was developed. Its quantitative performance was evaluated for confirmatory analysis of regulated lipophilic marine toxins in shellfish flesh according to Commission Decision 2002/657/EC. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX), and 13-desmethyl spirolide C (SPX-1) were quantified using matrix-matched calibration curves (MMS). For all compounds, the reproducibility ranged from 2.9 to 4.9 %, repeatability from 2.9 to 4.9 %, and recoveries from 82.9 to 113 % at the three different spiked levels. In addition, confirmatory identification of the compounds was effectively performed by the presence of a second diagnostic ion ((13)C). In conclusion, UHPLC-HR-Orbitrap MS permitted more accurate and faster detection of the target toxins than previously described LC-MS/MS methods. Furthermore, HRMS allows to retrospectively screen for many analogues and metabolites using its full-scan capabilities but also untargeted screening through the use of metabolomics software.

Understanding the effect of carbon status on stem diameter variations.

BACKGROUND: Carbon assimilation and leaf-to-fruit sugar transport are, along with plant water status, the driving mechanisms for fruit growth. An integrated comprehension of the plant water and carbon relationships is therefore essential to better understand water and dry matter accumulation. Variations in stem diameter result from an integrated response to plant water and carbon status and are as such a valuable source of information. METHODS: A mechanistic water flow and storage model was used to relate variations in stem diameter to phloem sugar loading and sugar concentration dynamics in tomato. The simulation results were compared with an independent model, simulating phloem sucrose loading at the leaf level based on photosynthesis and sugar metabolism kinetics and enabled a mechanistic interpretation of the 'one common assimilate pool' concept for tomato. KEY RESULTS: Combining stem diameter variation measurements and mechanistic modelling allowed us to distinguish instantaneous dynamics in the plant water relations and gradual variations in plant carbon status. Additionally, the model combined with stem diameter measurements enabled prediction of dynamic variables which are difficult to measure in a continuous and non-destructive way, such as xylem water potential and phloem hydrostatic potential. Finally, dynamics in phloem sugar loading and sugar concentration were distilled from stem diameter variations. CONCLUSIONS: Stem diameter variations, when used in mechanistic models, have great potential to continuously monitor and interpret plant water and carbon relations under natural growing conditions.

Comprehensive metabolomics reveals correlation between sophorolipid biosynthesis and autophagy.

Sophorolipids are biobased and biodegradable glycolipid surface-active agents contributing to the shift from petroleum to biobased surfactants, associated with clear environmental benefits. However, their production cost is currently too high to allow commercialisation. Therefore, a continuous sophorolipid production process was evaluated, i.e., a retentostat with an external filtration unit. Despite an initial increase in volumetric productivity, productivity eventually declined to almost 0 g L-1 h-1. Following comprehensive metabolomics on supernatant obtained from a standardised retentostat, we hypothesised exhaustion of the N-starvation-induced autophagy as the main mechanism responsible for the decline in bolaform sophorolipid productivity. Thirty-six metabolites that correlate with RNA/protein autophagy and high sophorolipid productivity were putatively identified. In conclusion, our results unveil a plausible cause of this bola sophorolipid productivity decline in an industrially relevant bioreactor set-up, which may thus impact majorly on future yeast biosurfactant regulation studies and the finetuning of bola sophorolipid production processes.

A multi-omics study to boost continuous bolaform sophorolipid production.

Biodegradable and biobased surface active agents are renewable and environmentally friendly alternatives to petroleum derived or oleochemical surfactants. However, they are accompanied by relatively high production costs. In this study, the aim was to reduce the production costs for an innovative type of microbial biosurfactant: bolaform sophorolipids, produced by the yeast Starmerella bombicola ΔsbleΔat. A novel continuous retentostat set-up was performed whereby continuous broth microfiltration retained the biomass in the bioreactor while performing an in situ product separation of bolaform sophorolipids. Although a mean volumetric productivity of 0.56 g L-1 h-1 was achieved, it was not possible to maintain this productivity, which collapsed to almost 0 g L-1 h-1. Therefore, two process adaptations were evaluated, a sequential batch strategy and a phosphate limitation alleviation strategy. The sequential batch set-up restored the mean volumetric productivity to 0.66 g L-1 h-1 for an additional 132 h but was again followed by a productivity decline. A similar result was obtained with the phosphate limitation alleviation strategy where a mean volumetric productivity of 0.54 g L-1 h-1 was reached, but a productivity decline was also observed. Whole genome variant analysis uncovered no evidence for genomic variations for up to 1306 h of retentostat cultivation. Untargeted metabolomics analysis identified 8-hydroxyguanosine, a biomarker for oxidative RNA damage, as a key metabolite correlating with high bolaform sophorolipid productivity. This study showcases the application of a retentostat to increase bolaform sophorolipid productivity and lays the basis of a multi-omics platform for in depth investigation of microbial biosurfactant production with S. bombicola.

The Bee Hemolymph Metabolome: A Window into the Impact of Viruses on Bumble Bees.

State-of-the-art virus detection technology has advanced a lot, yet technology to evaluate the impacts of viruses on bee physiology and health is basically lacking. However, such technology is sorely needed to understand how multi-host viruses can impact the composition of the bee community. Here, we evaluated the potential of hemolymph metabolites as biomarkers to identify the viral infection status in bees. A metabolomics strategy based on ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry was implemented. First, we constructed a predictive model for standardized bumble bees, in which non-infected bees were metabolically differentiated from an overt Israeli acute paralysis virus (IAPV) infection (R2Y = 0.993; Q2 = 0.906), as well as a covert slow bee paralysis virus (SBPV) infection (R2Y = 0.999; Q2 = 0.875). Second, two sets of potential biomarkers were identified, being descriptors for the metabolomic changes in the bee's hemolymph following viral infection. Third, the biomarker sets were evaluated in a new dataset only containing wild bees and successfully discriminated virus infection versus non-virus infection with an AUC of 0.985. We concluded that screening hemolymph metabolite markers can underpin physiological changes linked to virus infection dynamics, opening promising avenues to identify, monitor, and predict the effects of virus infection in a bee community within a specific environment.

Comprehensive polar metabolomics and lipidomics profiling discriminates the transformed from the non-transformed state in colon tissue and cell lines.

Colorectal cancer (CRC) is the fourth most lethal disease worldwide. Despite an urgent need for therapeutic advance, selective target identification in a preclinical phase is hampered by molecular and metabolic variations between cellular models. To foster optimal model selection from a translational perspective, we performed untargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry-based polar metabolomics and lipidomics to non-transformed (CCD841-CON and FHC) and transformed (HCT116, HT29, Caco2, SW480 and SW948) colon cell lines as well as tissue samples from ten colorectal cancer patients. This unveiled metabolic signatures discriminating the transformed from the non-transformed state. Metabolites involved in glutaminolysis, tryptophan catabolism, pyrimidine, lipid and carnitine synthesis were elevated in transformed cells and cancerous tissue, whereas those involved in the glycerol-3-phosphate shuttle, urea cycle and redox reactions were lowered. The degree of glutaminolysis and lipid synthesis was specific to the colon cancer cell line at hand. Thus, our study exposed pathways that are specifically associated with the transformation state and revealed differences between colon cancer cell lines that should be considered when targeting cancer-associated pathways.

Triangulation of microbial fingerprinting in anaerobic digestion reveals consistent fingerprinting profiles.

The anaerobic digestion microbiome has been puzzling us since the dawn of molecular methods for mixed microbial community analysis. Monitoring of the anaerobic digestion microbiome can either take place via a non-targeted holistic evaluation of the microbial community through fingerprinting or by targeted monitoring of selected taxa. Here, we compared four different microbial community fingerprinting methods, i.e., amplicon sequencing, metaproteomics, metabolomics and cytomics, in their ability to characterise the full-scale anaerobic digestion microbiome. Cytometric fingerprinting through cytomics reflects a, for anaerobic digestion, novel, single cell-based approach of direct microbial community fingerprinting by flow cytometry. Three different digester types, i.e., sludge digesters, digesters treating agro-industrial waste and dry anaerobic digesters, each reflected different operational parameters. The α-diversity analysis yielded inconsistent results, especially for richness, across the different methods. In contrast, ß-diversity analysis resulted in comparable profiles, even when translated into phyla or functions, with clear separation of the three digester types. In-depth analysis of each method's features i.e., operational taxonomic units, metaproteins, metabolites, and cytometric traits, yielded certain similar features, yet, also some clear differences between the different methods, which was related to the complexity of the anaerobic digestion process. In conclusion, cytometric fingerprinting through flow cytometry is a reliable, fast method for holistic monitoring of the anaerobic digestion microbiome, and the complementary identification of key features through other methods could give rise to a direct interpretation of anaerobic digestion process performance.

Untargeted Metabolomics Reveals Elevated L-Carnitine Metabolism in Pig and Rat Colon Tissue Following Red Versus White Meat Intake.

SCOPE: The consumption of red and processed meat, and not white meat, associates with the development of various Western diseases such as colorectal cancer and type 2 diabetes. This work aims at unraveling novel meat-associated mechanisms that are involved in disease development. METHODS AND RESULTS: A non-hypothesis driven strategy of untargeted metabolomics is applied to assess colon tissue from rats (fed a high dose of beef vs. white meat) and from pigs (fed red/processed meat vs. white meat), receiving a realistic human background diet. An increased carnitine metabolism is observed, which is reflected by higher levels of acylcarnitines and 3-dehydroxycarnitine (rats and pigs) and trimethylamine-N-oxide (rats). While 3-dehydroxycarnitine is higher in HT29 cells, incubated with colonic beef digests, acylcarnitine levels are reduced. This suggests an altered response from colon cancer cell line towards meat-induced oxidative stress. Moreover, metabolic differences between rat and pigs are observed in N-glycolylneuraminic acid incorporation, prostaglandin, and fatty acid synthesis. CONCLUSION: This study demonstrates elevated (acyl)carnitine metabolism in colon tissue of animals that follow a red meat-based diet, providing mechanistic insights that may aid in explaining the nutritional-physiological correlation between red/processed meat and Western diseases.

Metabolic Fingerprinting of Feces from Calves, Subjected to Gram-Negative Bacterial Endotoxin.

Gram-negative bacteria have a well-known impact on the disease state of neonatal calves and their mortality. This study was the first to implement untargeted metabolomics on calves' fecal samples to unravel the effect of Gram-negative bacterial endotoxin lipopolysaccharide (LPS). In this context, calves were challenged with LPS and administered with fish oil, nanocurcumin, or dexamethasone to evaluate treatment effects. Ultra-high-performance liquid-chromatography high-resolution mass spectrometry (UHPLC-HRMS) was employed to map fecal metabolic fingerprints from the various groups before and after LPS challenge. Based on the generated fingerprints, including 9650 unique feature ions, significant separation according to LPS group was achieved through orthogonal partial least squares discriminant analysis (Q2 of 0.57 and p-value of 0.022), which allowed the selection of 37 metabolites as bacterial endotoxin markers. Tentative identification of these markers suggested that the majority belonged to the subclass of the carboxylic acid derivatives-amino acids, peptides, and analogs-and fatty amides, with these subclasses playing a role in the metabolism of steroids, histidine, glutamate, and folate. Biological interpretations supported the revealed markers' potential to aid in disease diagnosis, whereas beneficial effects were observed following dexamethasone, fish oil, and nanocurcumin treatment.