Untargeted Plant Metabolomics: Evaluation of Lyophilization as a Sample Preparation Technique.

Lyophilization is a common method used for stabilizing biological samples prior to storage or to concentrate extracts. However, it is possible that this process may alter the metabolic composition or lead to the loss of metabolites. In this study, the performance of lyophilization is investigated in the example of wheat roots. To this end, native and 13C-labelled, fresh or already lyophilized root samples, and (diluted) extracts with dilution factors up to 32 and authentic reference standards were investigated. All samples were analyzed using RP-LC-HRMS. Results show that using lyophilization for the stabilization of plant material altered the metabolic sample composition. Overall, 7% of all wheat metabolites detected in non-lyophilized samples were not detected in dried samples anymore, and up to 43% of the remaining metabolites exhibited significantly increased or decreased abundances. With respect to extract concentration, less than 5% of the expected metabolites were completely lost by lyophilization and the recovery rates of the remaining metabolites were slightly reduced with increasing concentration factors to an average of 85% at an enrichment factor of 32. Compound annotation did not indicate specific classes of wheat metabolites to be affected.

Biosynthesis of the Isocoumarin Derivatives Fusamarins is Mediated by the PKS8 Gene Cluster in Fusarium.

Fusarium mangiferae causes the mango malformation disease (MMD) on young mango trees and seedlings resulting in economically significant crop losses. In addition, F. mangiferae produces a vast array of secondary metabolites (SMs), including mycotoxins that may contaminate the harvest. Their production is tightly regulated at the transcriptional level. Here, we show that lack of the H3 K9-specific histone methyltransferase, FmKmt1, influences the expression of the F. mangiferae polyketide synthase (PKS) 8 (FmPKS8), a so far cryptic PKS. By a combination of reverse genetics, untargeted metabolomics, bioinformatics and chemical analyses including structural elucidation, we determined the FmPKS8 biosynthetic gene cluster (BGC) and linked its activity to the production of fusamarins (FMN), which can be structurally classified as dihydroisocoumarins. Functional characterization of the four FMN cluster genes shed light on the biosynthetic pathway. Cytotoxicity assays revealed moderate toxicities with IC50 values between 1 and 50 µM depending on the compound.

Improving Properties of Starch-Based Adhesives with Carboxylic Acids and Enzymatically Polymerized Lignosulfonates.

A novel strategy for improving wet resistance and bonding properties of starch-based adhesives using enzymatically polymerized lignosulfonates and carboxylic acids as additives was developed. Therefore, lignosulfonates were polymerized by laccase to a molecular weight of 750 kDa. Incorporation of low concentrations (up to 1% of the starch weight) of 1,2,3,4-butanetetracarboxylic acid (BTCA) led to further improvement on the properties of the adhesives, while addition of greater amounts of BTCA led to a decrease in the properties measured due to large viscosity increases. Great improvements in wet-resistance from 22 to 60 min and bonding times (from 30 to 20 s) were observed for an adhesive containing 8% enzymatically polymerized lignin and 1% BTCA. On the other hand, the addition of citric acid (CA) deteriorated the properties of the adhesives, especially when lignosulfonate was present. In conclusion, this study shows that the addition of the appropriate amount of enzymatically polymerized lignosulfonates together with carboxylic acids (namely BTCA) to starch-based adhesives is a robust strategy for improving their wet resistance and bonding times.

Gramiketides, Novel Polyketide Derivatives of Fusarium graminearum, Are Produced during the Infection of Wheat.

The plant pathogen Fusarium graminearum is a proficient producer of mycotoxins and other in part still unknown secondary metabolites, some of which might act as virulence factors on wheat. The PKS15 gene is expressed only in planta, so far hampering the identification of an associated metabolite. Here we combined the activation of silent gene clusters by chromatin manipulation (kmt6) with blocking the metabolic flow into the competing biosynthesis of the two major mycotoxins deoxynivalenol and zearalenone. Using an untargeted metabolomics approach, two closely related metabolites were found in triple mutants (kmt6 tri5 pks4,13) deficient in production of the major mycotoxins deoxynivalenol and zearalenone, but not in strains with an additional deletion in PKS15 (kmt6 tri5 pks4,13 pks15). Characterization of the metabolites, by LC-HRMS/MS in combination with a stable isotope-assisted tracer approach, revealed that they are likely hybrid polyketides comprising a polyketide part consisting of malonate-derived acetate units and a structurally deviating part. We propose the names gramiketide A and B for the two metabolites. In a biological experiment, both gramiketides were formed during infection of wheat ears with wild-type but not with pks15 mutants. The formation of the two gramiketides during infection correlated with that of the well-known virulence factor deoxynivalenol, suggesting that they might play a role in virulence.

An Organic Chemist's Guide to Mediated Laccase Oxidation.

Laccases are oxidases that only require O2 as a terminal oxidant. Thus, they provide an attractive green alternative to established alcohol oxidation protocols. However, laccases typically require catalytic amounts of mediator molecules to serve as electron shuttles between the enzyme and desired substrate. Consequently, laccase-mediator systems are defined by a multitude of parameters such as, e. g., the choice of laccase and mediator, the respective concentrations, pH, and the oxygen source. This complexity and a perceived lack of comparable data throughout literature represent an entry burden into this field. To provide a solid starting point, particularly for organic chemists, we herein provide a time-resolved, quantitative laccase and mediator screening based on the oxidation of anis alcohol as model reaction. We measured the redox potentials of mediators under the reaction conditions to relate them to their performance. Lastly, for particularly efficient laccase-mediator pairs, we screened important reaction parameters, resulting in an optimized setup for mediator-assisted laccase catalyzed oxidations.

Heterogeneous multimeric metabolite ion species observed in LC-MS based metabolomics data sets.

Covalent or non-covalent heterogeneous multimerization of molecules associated with extracts from biological samples analyzed via LC-MS are quite difficult to recognize/annotate and therefore the prevalence of multimerization remains largely unknown. In this study, we utilized 13C labeled and unlabeled Pichia pastoris extracts to recognize heterogeneous multimers. More specifically, between 0.8% and 1.5% of the biologically-derived features detected in our experiments were confirmed to be heteromers, about half of which we could successfully annotate with monomeric partners. Interestingly, we found specific chemical classes such as nucleotides to disproportionately contribute to heteroadducts. Furthermore, we compiled these compounds into the first MS/MS library that included data from heteromultimers to provide a starting point for other labs to improve the annotation of such ions in other metabolomics data sets. Then, the detected heteromers were also searched in publicly accessible LC-MS datasets available in Metabolights, Metabolomics WB and GNPS/MassIVE to demonstrate that these newly annotated ions are also relevant to other public datasets. Furthermore, in additional datasets (Triticum aestivum, Fusarium graminearum, and Trichoderma reesei) our developed workflow also detected 0.5%-4.9% of metabolite features to originate from heterodimers, demonstrating heteroadducts to be present in metabolomics studies at a low percentage.

PeakBot: machine-learning-based chromatographic peak picking.

MOTIVATION: Chromatographic peak picking is among the first steps in data processing workflows of raw LC-HRMS datasets in untargeted metabolomics applications. Its performance is crucial for the holistic detection of all metabolic features as well as their relative quantification for statistical analysis and metabolite identification. Random noise, non-baseline separated compounds and unspecific background signals complicate this task. RESULTS: A machine-learning-based approach entitled PeakBot was developed for detecting chromatographic peaks in LC-HRMS profile-mode data. It first detects all local signal maxima in a chromatogram, which are then extracted as super-sampled standardized areas (retention-time versus m/z). These are subsequently inspected by a custom-trained convolutional neural network that forms the basis of PeakBot's architecture. The model reports if the respective local maximum is the apex of a chromatographic peak or not as well as its peak center and bounding box. In training and independent validation datasets used for development, PeakBot achieved a high performance with respect to discriminating between chromatographic peaks and background signals (accuracy of 0.99). For training the machine-learning model a minimum of 100 reference features are needed to learn their characteristics to achieve high-quality peak-picking results for detecting such chromatographic peaks in an untargeted fashion. PeakBot is implemented in python (3.8) and uses the TensorFlow (2.5.0) package for machine-learning related tasks. It has been tested on Linux and Windows OSs. AVAILABILITY AND IMPLEMENTATION: The package is available free of charge for non-commercial use (CC BY-NC-SA). It is available at https://github.com/christophuv/PeakBot. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Enzymatic synthesis of wet-resistant lignosulfonate-starch adhesives.

This work describes a new method for improving the properties, mainly the wet-resistance, of starch-based adhesives using enzymatically polymerized lignosulfonates. A correlation of viscosity with molecular weight was found, allowing simple control of enzymatic polymerization of lignosulfonates. Incorporation of lignosulfonates polymerized from 29 kDa to > 4500 kDa using laccase led to a considerable increase in wet-resistance (from 15 to 20 min for the laminating glue and from 150 to 1200 min for the bag glue) while not affecting (for the laminating glue) or even improving the bonding time (from 80 to 60 s for the bag glue). Finally, the effect of active laccase in the final adhesive was investigated by enzymatic inactivation using NaN3 before formulation of the glue, as well as by extra laccase addition. In conclusion, this study shows that enzymatically polymerized lignosulfonate is a robust strategy for improving wet resistance of starch-based adhesives.

CPExtract, a Software Tool for the Automated Tracer-Based Pathway Specific Screening of Secondary Metabolites in LC-HRMS Data.

The use of stable isotopically labeled tracers is a long-proven way of specifically detecting and tracking derived metabolites through a metabolic network of interest. While the recently developed stable isotope-assisted methods and associated, supporting data analysis tools have greatly improved untargeted metabolomics approaches, no software tool is currently available that allows us to automatically and flexibly search liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) chromatograms for user-definable isotopolog patterns expected for the metabolism of labeled tracer substances. Here, we present Custom Pattern Extract (CPExtract), a versatile software tool that allows for the first time the high-throughput search for user-defined isotopolog patterns in LC-HRMS data. The patterns can be specified via a set of rules including the presence or absence of certain isotopologs, their relative intensity ratios as well as chromatographic coelution. Each isotopolog pattern satisfying the respective rules is verified on an MS scan level and also in the chromatographic domain. The CPExtract algorithm allows the use of both labeled tracer compounds in nonlabeled biological samples as well as a reversed tracer approach, employing nonlabeled tracer compounds along with globally labeled biological samples. In a proof-of-concept study, we searched for metabolites specifically arising from the malonate pathway of the filamentous fungi Fusarium graminearum and Trichoderma reesei. 1,2,3-13C3-malonic acid diethyl ester and native malonic acid monomethyl ester were used as tracers. We were able to reliably detect expected fatty acids and known polyketides. In addition, up to 46 and 270 further, unknown metabolites presumably including novel polyketides were detected in the F. graminearum and T. reesei culture samples, respectively, all of which exhibited the user-predicted isotopolog patterns originating from the malonate tracer incorporation. The software can be used for every conceivable tracer approach. Furthermore, the rule sets can be easily adapted or extended if necessary. CPExtract is available free of charge for noncommercial use at https://metabolomics-ifa.boku.ac.at/CPExtract.

The Comprehensive and Reliable Detection of Secondary Metabolites in Trichoderma reesei: A Tool for the Discovery of Novel Substances.

A method based on reversed phase high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (RP-HPLC-ESI-HRMS) for the comprehensive and reliable detection of secondary metabolites of Trichoderma reesei cultured in synthetic minimal liquid medium is presented. A stable isotope-assisted (SIA) workflow is used, which allows the automated, comprehensive extraction of truly fungal metabolite-derived LC-MS signals from the acquired chromatographic data. The subsequent statistical data analysis and a typical outcome of such a metabolomics data evaluation are shown by way of example in a previously published study on the influence of the pleiotropic regulator transcription factor Xylanase promoter binding protein 1 (Xpp1) in T. reesei on secondary metabolism.

Risk analysis for intraoperative liver surgery.

Hepatic vessel structure is very important to ensure the blood supply of the liver tissue. Therefore the knowledge of the hepatic vessel system is indispensable in liver surgery planning, for example before performing a liver resection. The purpose of this paper is to present an easy to use and fast method concerning hepatic vessel segmentation and risk analysis, which is intended to be applicable in clinical routine.

Effect of cardiac resynchronization therapy on the incidence of electrical storm.

BACKGROUND: Hemodynamic improvement from biventricular pacing is well documented; however, its electrophysiologic effects have not been systematically studied. In this study, incidence and risk factors for electrical storm (ES) were investigated in 729 ICD and biventricular defibrillator (CRT-D) heart failure patients. METHODS: 168 consecutive CRT-D and 561 ICD patients were retrospectively analyzed for the occurrence of VT/VF and predisposing factors. Electrical storm was defined as ventricular tachycardia or fibrillation ≥3 times during 24 h. Mean follow-up was 41 months. RESULTS: In 168 CRT-D patients only one patient experienced electrical storm compared to 39 patients out of 561 ICD patients (0.6% vs. 7%, p<0.01). 33% of the patients with electrical storm died within one year. In the CRT-D group 81 patients (48%) developed VT or VF and received at least one appropriate therapy, compared to 281 patients (50%) in the ICD group. Mean ejection fraction was 21.7% in the CRT-D group and 34.7% (p<0.01) in the ICD group. Stratifying the patients according to primary or secondary prevention and ejection fraction demonstrated that VT/VF clusters were significantly associated with ICD indication for secondary prevention, previous myocardial infarction and LVEF<30%. CONCLUSION: The development of electrical storm is accompanied with a highly increased mortality risk even if an ICD/CRT-D is implanted. In CRT-D patients electrical storm is much less common than in ICD patients. Secondary prevention and ejection fraction<30% are predictors of electrical storm. Beside hemodynamic improvements cardiac resynchronization therapy may reduce the arrhythmia burden in heart failure patients.