Investigating the Metabolism of Plants Germinated in Heavy Water, D2O, and H218O-Enriched Media Using High-Resolution Mass Spectrometry.

Mass spectrometry has been an essential technique for the investigation of the metabolic pathways of living organisms since its appearance at the beginning of the 20th century. Due to its capability to resolve isotopically labeled species, it can be applied together with stable isotope tracers to reveal the transformation of particular biologically relevant molecules. However, low-resolution techniques, which were used for decades, had limited capabilities for untargeted metabolomics, especially when a large number of compounds are labelled simultaneously. Such untargeted studies may provide new information about metabolism and can be performed with high-resolution mass spectrometry. Here, we demonstrate the capabilities of high-resolution mass spectrometry to obtain insights on the metabolism of a model plant, Lepidium sativum, germinated in D2O and H218O-enriched media. In particular, we demonstrated that in vivo labeling with heavy water helps to identify if a compound is being synthesized at a particular stage of germination or if it originates from seed content, and tandem mass spectrometry allows us to highlight the substructures with incorporated isotope labels. Additionally, we found in vivo labeling useful to distinguish between isomeric compounds with identical fragmentation patterns due to the differences in their formation rates that can be compared by the extent of heavy atom incorporation.

New Pseudomonas Bacterial Strains: Biological Activity and Characteristic Properties of Metabolites.

This paper investigates the antagonistic and plant growth promotion activity of the new indigenous bacteria antagonist strains P. chlororaphis BZR 245-F and Pseudomonas sp. BZR 523-2. It was found that on the 10th day of cultivation, BZR 245-F and BZR 523-2 exhibit an antagonistic activity against F. graminearum at the level of 59.6% and 15.1% and against F. oxysporum var. orthoceras at the level of 50.2% and 8.9%, respectively. Furthermore, the BZR 523-2 strain stimulated the growth of winter wheat seedlings more actively than the BZR 245-F strain. When processing seeds of winter wheat, Pseudomonas sp. BZR 523-2 indicators were higher than in the control: plant height increased by 10.3%, and root length increased by 18.6%. The complex characteristic properties of the metabolite were studied by bioautography and HPLC-MS. Bioautography proved the antifungal activity of phenazine nature compounds synthesized by the new bacterial strains. We qualitatively and quantitatively analyzed them by HPLC-MS analysis of the strain sample metabolites. In the BZR 245-F sample, we found more phenazine compounds of various types. Their total phenazine concentration in the BZR 245-F was more than five times greater than in the BZR 523-2. We defined crucial differences in the quantitative content of the other metabolites. Despite the difference between new indigenous bacteria antagonist strains, they can be used as producers of effective biopesticides for sustainable agriculture management.

Untargeted Lipidomics after D2O Administration Reveals the Turnover Rate of Individual Lipids in Various Organs of Living Organisms.

The administration of low doses of D2O to living organisms was used for decades for the investigation of metabolic pathways and for the measurement of the turnover rate for specific compounds. Usually, the investigation of the deuterium uptake in lipids is performed by measuring the deuteration level of the palmitic acid residue using GC-MS instruments, and to our knowledge, the application of the modern untargeted LC-MS/MS lipidomics approaches was only reported a few times. Here, we investigated the deuterium uptake for >500 lipids for 13 organs and body liquids of mice (brain, lung, heart, liver, kidney, spleen, plasma, urine, etc.) after 4 days of 100% D2O administration. The maximum deuteration level was observed in the liver, plasma, and lung, while in the brain and heart, the deuteration level was lower. Using MS/MS, we demonstrated the incorporation of deuterium in palmitic and stearic fragments in lipids (PC, PE, TAG, PG, etc.) but not in the corresponding free forms. Our results were analyzed based on the metabolic pathways of lipids.

Simple In Vitro 18O Labeling for Improved Mass Spectrometry-Based Drug Metabolites Identification: Deep Drug Metabolism Study.

The identification of drug metabolites formed with different in vitro systems by HPLC-MS is a standard step in preclinical research. In vitro systems allow modeling of real metabolic pathways of a drug candidate. Despite the emergence of various software and databases, identification of compounds is still a complex task. Measurement of the accurate mass, correlation of chromatographic retention times and fragmentation spectra are often insufficient for identification of compounds especially in the absence of reference materials. Metabolites can "slip under the nose", since it is often not possible to reliably confirm that a signal belongs to a metabolite and not to other compounds in complex systems. Isotope labeling has proved to be a tool that aids in small molecule identification. The introduction of heavy isotopes is done with isotope exchange reactions or with complicated synthetic schemes. Here, we present an approach based on the biocatalytic insertion of oxygen-18 isotope under the action of liver microsomes enzymes in the presence of 18O2. Using the local anesthetic bupivacaine as an example, more than 20 previously unknown metabolites were reliably discovered and annotated in the absence of the reference materials. In combination with high-resolution mass spectrometry and modern methods of mass spectrometric metabolism data processing, we demonstrated the ability of the proposed approach to increase the degree of confidence in interpretating metabolism data.

Red light-emitting short Mango-based system enables tracking a mycobacterial small noncoding RNA in infected macrophages.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

Unprecedented Coordination-Induced Bright Red Emission from Group 12 Metal-Bound Triarylazoimidazoles.

Arylazoimidazoles are important dyes which were intensively studied in the past. In contrast, triarylazoimidazoles (derivatives which carry aryl substituents at the imidazole core) received almost no attention in the scientific literature. Here, we report a new family of simple and easily accessible triarylazoimidazole-group 12 metal complexes, which feature highly efficient photo-luminescence emission (Φ up to 0.44). Novel compounds exhibit bright red emission in solution, which could be excited with a visible light.