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Desiccating and germinating seeds are particularly prone to oxidative stress. Glutathione (GSH) is one of the most abundant and ubiquitous antioxidants in plants. To better understand GSH's role in developing and germinating seeds, wildtype (WT) and a line of seed antisense for glutathione reductase 2 (anGR2), one of two glutathione reductase (GR) genes characterized in the genetic model plant Arabidopsis thaliana, were compared. GSH levels in maturing and germinating seeds were measured by high performance liquid chromatography (HPLC) and GR activity by native Polyacrylamide gel electrophoresis (PAGE). Natural and accelerated aging and germination at high temperature and under water stress were compared for WT and anGR2 seeds. Total GSH pools were two-fold less in anGR2 than in WT after three hours of imbibition. Under high temperature and osmotic stress, germination percentages were lower in anGR2, suggesting a specific relationship between GSH metabolism and stress defense mechanisms early in germination. Germination of anGR2 seeds declined while that of WT seeds increased slightly, during nine months of conventional storage. The relationship between GR2 transcript and activity levels during early imbibition and total GSH pools suggest a key role for GR2 in protection against environmental stress. This study shows that GSH metabolism is one factor that determines the stress tolerance of germinating Arabidopsis seeds. [ABSTRACT FROM AUTHOR]

Abstract Background Rosaceae include numerous economically important and morphologically diverse species. Comparative mapping between the member species in Rosaceae have indicated some level of synteny. Recently the whole genome of three crop species, peach, apple and strawberry, which belong to different genera of the Rosaceae family, have been sequenced, allowing in-depth comparison of these genomes. Results Our analysis using the whole genome sequences of peach, apple and strawberry identified 1399 orthologous regions between the three genomes, with a mean length of around 100 kb. Each peach chromosome showed major orthology mostly to one strawberry chromosome, but to more than two apple chromosomes, suggesting that the apple genome went through more chromosomal fissions in addition to the whole genome duplication after the divergence of the three genera. However, the distribution of contiguous ancestral regions, identified using the multiple genome rearrangements and ancestors (MGRA) algorithm, suggested that the Fragaria genome went through a greater number of small scale rearrangements compared to the other genomes since they diverged from a common ancestor. Using the contiguous ancestral regions, we reconstructed a hypothetical ancestral genome for the Rosaceae 7 composed of nine chromosomes and propose the evolutionary steps from the ancestral genome to the extant Fragaria, Prunus and Malus genomes. Conclusion Our analysis shows that different modes of evolution may have played major roles in different subfamilies of Rosaceae. The hypothetical ancestral genome of Rosaceae and the evolutionary steps that lead to three different lineages of Rosaceae will facilitate our understanding of plant genome evolution as well as have a practical impact on knowledge transfer among member species of Rosaceae.

Recurrent urinary tract infections (rUTIs) are a major clinical challenge in postmenopausal women and their increasing prevalence underscores the need to define interactions between the host and the urinary microbiome that may underlie rUTI susceptibility. A body of work has identified the taxonomic profile of the female urinary microbiome associate with aging, menopause, and urinay disease. However, how this microbial community engages with the host niche, including the local biochemical environment of the urogenital tract, in health and disease is yet to be fully defined. This study directly assesses differences in the biochemical environment of the urine, or biochemical ecology, associated with recurrent urinary tract infection (UTI) and defines a microbe-metabolite association network of the female urinary microbiome. By integrating metagenomic and metabolomic data collected from a controlled cohort of women with rUTI, we find that distinct metabolites, such as methionine sulfoxide (Met-SO) and trimethylamine oxide (TMAO), are associated with differences in urinary microbiome diversity. We observe associations between microbial and biochemical beta diversity and unique metabolic networks of uropathogenic Escherichia coli and uroprotective Lactobacillus species, highlighting potential metabolite-driven ecological shifts that may influence UTI susceptibility. We identify a urinary lipid signature of active rUTI that can accurately distinguish (AUC = 0.987) cases controls. Finally, using time-to-relapse data we identify deoxycholic acid (DCA) as a new prognostic indicator for rUTI recurrence. Together these findings suggest that systemic metabolic processes may influence susceptibility, opening new avenues for therapeutic intervention and the development of more accurate diagnostic and prognostic to improve patient outcomes.

This Perspective covers discovery and mechanistic aspects as well as initial applications of novel ionization processes for use in mass spectrometry that guided us in a series of subsequent discoveries, instrument developments, and commercialization. Vacuum matrix-assisted ionization on an intermediate pressure matrix-assisted laser desorption/ionization source without the use of a laser, high voltages, or any other added energy was simply unbelievable, at first. Individually and as a whole, the various discoveries and inventions started to paint, inter alia , an exciting new picture and outlook in mass spectrometry from which key developments grew that were at the time unimaginable, and continue to surprise us in its simplistic preeminence. We, and others, have demonstrated exceptional analytical utility. Our current research is focused on how best to understand, improve, and use these novel ionization processes through dedicated platforms and source developments. These ionization processes convert volatile and nonvolatile compounds from solid or liquid matrixes into gas-phase ions for analysis by mass spectrometry using, e.g. , mass-selected fragmentation and ion mobility spectrometry to provide accurate, and sometimes improved, mass and drift time resolution. The combination of research and discoveries demonstrated multiple advantages of the new ionization processes and established the basis of the successes that lead to the Biemann Medal and this Perspective. How the new ionization processes relate to traditional ionization is also presented, as well as how these technologies can be utilized in tandem through instrument modification and implementation to increase coverage of complex materials through complementary strengths.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making., (© 2024 John Wiley & Sons Ltd.)

Eight Ribes magellanicum collections from three different places in southern Patagonia were compared for content of different groups of phenolics, antioxidant capacity and inhibition of enzymes related to metabolic syndrome (α-amylase, α-glucosidase and pancreatic lipase). The sample with the highest antioxidant capacity was assessed for glutathione (GSH) synthesis stimulation in human gastric adenocarcinoma (AGS) cells. The chemical profile was determined by high performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS) and the main phenolics were quantified. The samples from Navarino Island and Reserva Nacional Magallanes showed higher content of anthocyanins and caffeoylquinic acid, with better activity towards α-glucosidase and antioxidant capacity. A sample from Omora (Navarino Island), significantly increased intracellular GSH content in AGS cells. Some 70 compounds were identified in the fruit extracts by HPLC-MS/MS. The glucoside and rutinoside from delphinidin and cyanidin and 3-caffeoylquinic acid were the main compounds. Different chemical profiles were found according to the collection places., Competing Interests: The authors declare that they have no known competing financial interests., (© 2024 The Authors. Published by Elsevier Ltd.)

Glycosaminoglycans (GAGs) are linear, negatively charged polysaccharides composed of repeating disaccharide units of uronic acid and amino sugars. The luminal surface of the bladder epithelium is coated with a GAG layer. These urothelial GAGs are thought to provide a protective barrier and serve as a potential interaction site with the urinary microbiome (urobiome). Previous studies have profiled urinary GAG composition in mixed cohorts, but the urinary GAG composition in postmenopausal women remains undefined. To investigate the relationship between GAGs and recurrent urinary tract infection (rUTI), we profiled urinary GAGs in a controlled cohort of postmenopausal women. We found that chondroitin sulfate (CS) is the major urinary GAG in postmenopausal women and that urinary CS was elevated in women with active rUTI. We also associated urinary GAGs with urobiome composition and identified bacterial species that significantly associated with urinary GAG concentration. Corynebacterium amycolatum , Porphyromonas somerae , and Staphylococcus pasteuri were positively associated with heparin sulfate or hyaluronic acid, and bacterial species associated with vaginal dysbiosis were negatively correlated with urinary CS. Altogether, this work defines changes in urinary GAG composition associated with rUTI and identifies new associations between urinary GAGs and the urobiome that may play a role in rUTI pathobiology.

Postmenopausal women are severely affected by recurrent urinary tract infection (rUTI). The urogenital microbiome is a key component of the urinary environment. However, changes in the urogenital microbiome underlying rUTI susceptibility are unknown. Here, we perform shotgun metagenomics and advanced culture on urine from a controlled cohort of postmenopausal women to identify urogenital microbiome compositional and function changes linked to rUTI susceptibility. We identify candidate taxonomic biomarkers of rUTI susceptibility in postmenopausal women and an enrichment of lactobacilli in postmenopausal women taking estrogen hormone therapy. We find robust correlations between Bifidobacterium and Lactobacillus and urinary estrogens in women without urinary tract infection (UTI) history. Functional analyses reveal distinct metabolic and antimicrobial resistance gene (ARG) signatures associated with rUTI. Importantly, we find that ARGs are enriched in the urogenital microbiomes of women with rUTI history independent of current UTI status. Our data suggest that rUTI and estrogen shape the urogenital microbiome in postmenopausal women., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Liquid chromatography-mass spectrometry (LC-MS) provides one of the most popular platforms for untargeted plant lipidomics analysis (Shulaev and Chapman, Biochim Biophys Acta 1862(8):786-791, 2017; Rupasinghe and Roessner, Methods Mol Biol 1778:125-135, 2018; Welti et al., Front Biosci 12:2494-506, 2007; Shiva et al., Plant Methods 14:14, 2018). We have developed SimLipid software in order to streamline the analysis of large-volume datasets generated by LC-MS-based untargeted lipidomics methods. SimLipid contains a customizable library of lipid species; graphical user interfaces (GUIs) for visualization of raw data; the identified lipid molecules and their associated mass spectra annotated with fragment ions and parent ions; and detailed information of each identified lipid species all in a single workbench enabling users to rapidly review the results by examining the data for confident identifications of lipid molecular species. In this chapter, we present the functionality of the software and workflow for automating large-scale LC-MS-based untargeted lipidomics profiling., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Lipids play an essential role in plants, and historically manipulating their levels and composition has been an important target for metabolic engineering. A variety of analytical techniques, many based on mass spectrometry, have been utilized for lipid profiling, but the analysis of complex lipid mixtures still poses significant analytical challenges. Recent advances in technology have revived the supercritical fluid chromatography (SFC) as a promising separation technique for lipid analysis. Utilization of sub-2-μm particle columns improves the separation efficiency and robustness of the SFC systems. The combination of SFC with sub-2-μm particle separation, commonly referred as ultra-performance convergence chromatography, has been successfully used for separation of both polar and neutral lipids. In this chapter, we present a simple method for lipid class separation using Sub-2-μm particle CO 2 -based chromatography coupled to mass spectrometry. The supercritical fluid chromatography methodology is flexible and can be altered to provide greater retention and separation of lipid classes or individual lipids within class., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Lipids play an important role in the energy storage, cellular signaling, and pathophysiology of diseases such as cancer, neurodegenerative diseases, infections, and diabetes. Due to high importance of diverse lipid classes in human health and disease, manipulating lipid abundance and composition is an important target for metabolic engineering. The extreme structural diversity of lipids in real biological samples is challenging for analytical techniques due to large difference in physicochemical properties of individual lipid species. This chapter describes lipidomic analysis of large sample sets requiring reliable and robust methodology. Rapid and robust methods facilitate the support of longitudinal studies allowing the transfer of methodology between laboratories. We describe a high-throughput reversed-phase LC-MS methodology using Ultra Performance Liquid Chromatography (UPLC ® ) with charged surface hybrid technology and accurate mass detection for high-throughput non-targeted lipidomics. The methodology showed excellent specificity, robustness, and reproducibility for over 100 LC-MS injections., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Discovery-driven comparative proteomics employing the bottom-up strategy with label-free quantification on high-resolution mass analyzers like an Orbitrap in a hybrid instrument has the capacity to reveal unique biological processes in the context of plant metabolic engineering. However, proteins are very heterogeneous in nature with a wide range of expression levels, and overall coverage may be suboptimal regarding both the number of protein identifications and sequence coverage of the identified proteins using conventional data-dependent acquisitions without sample fractionation before online nanoflow liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS). In this chapter, we detail a simple and robust method employing high-pH reversed-phase (HRP) peptide fractionation using solid-phase extraction cartridges for label-free proteomic analyses. Albeit HRP fractionation separates peptides according to their hydrophobicity like the subsequent nanoflow gradient reversed-phased LC relying on low pH mobile phase, the two methods are orthogonal. Presented here as a protocol with yeast (Saccharomyces cerevisiae) as a frequently used model organism and hydrogen peroxide to exert cellular stress and survey its impact compared to unstressed control as an example, the described workflow can be adapted to a wide range of proteome samples for applications to plant metabolic engineering research., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Gas chromatography coupled to electron ionization (EI) quadrupole mass spectrometry (GC-MS) is currently one of the most developed and robust metabolomics technologies. This approach allows for simultaneous measurements of large number of chemically diverse compounds including organic acids, amino acids, sugars, sugar alcohols, aromatic amines, and fatty acids. Untargeted GC-MS profiling based on full scan data acquisition requires complicated raw data processing and sometime provides ambiguous metabolite identifications. Targeted analysis using GC-MS/MS can provide better specificity, increase sensitivity, and simplify data processing and compound identification but wider application of targeted GC-MS/MS approach in metabolomics is hampered by the lack of extensive databases of MRM transitions for non-derivatized and derivatized endogenous metabolites. The focus of this chapter is the automation of GC-MS/MS method development which makes it feasible to develop quantitative methods for several hundred metabolites and use this strategy for plant metabolomics applications., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Analysis of volatile compounds in fruits and plants can be a challenging task as they present in a large amount with structural diversity and high aroma threshold, the information on molecular ion can be very useful for compound identification. Electron ionization gas-chromatography-mass spectrometry (EI-GC-MS) which is widely used for the analysis of plant volatiles has a certain limitation providing the limited capability to characterize novel metabolites in a complex biological matrix due to hard fragmentation level. Atmospheric pressure ionization using APGC source in combination with high-resolution time-of-flight mass spectrometry (TOF-MS) provides an excellent combination of GC with high-resolution mass spectrometry. The APGC-MS approach provides several advantages over the conventional EI and CI based GC-MS techniques in metabolomics studies due to highly reduced fragmentation, which preserves molecular ion, and accurate mass measurement by HRMS allows to deduce the elemental composition of the volatile compounds. Moreover, the use of MS E mode provides spectral similarity to EI in high-energy mode which can be used for the further confirmation of metabolite identity. We describe an APGC-MS-based untargeted metabolomics approach with a case study of grape volatile compounds and the development of a spectral library for metabolite identification., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

The early signaling events involved in oxidant recognition and triggering of oxidant-specific defense mechanisms to counteract oxidative stress still remain largely elusive. Our discovery driven comparative proteomics analysis revealed unique early signaling response of the yeast Saccharomyces cerevisiae on the proteome level to oxidants with a different mechanism of action as early as 3 min after treatment with four oxidants, namely H 2 O 2 , cumene hydroperoxide (CHP), and menadione and diamide, when protein abundances were compared using label-free quantification relying on a high-resolution mass analyzer (Orbitrap). We identified significant regulation of 196 proteins in response to H 2 O 2 , 569 proteins in response to CHP, 369 proteins in response to menadione and 207 proteins in response to diamide. Only 17 proteins were common across all treatments, but several more proteins were shared between two or three oxidants. Pathway analyses revealed that each oxidant triggered a unique signaling mechanism associated with cell survival and repair. Signaling pathways mostly regulated by oxidants were Ran, TOR, Rho, and eIF2. Furthermore, each oxidant regulated these pathways in a unique way indicating specificity of response to oxidants having different modes of action. We hypothesize that interplay of these signaling pathways may be important in recognizing different oxidants to trigger different downstream MAPK signaling cascades and to induce specific responses.

Precision medicine is rapidly emerging as a strategy to tailor medical treatment to a small group or even individual patients based on their genetics, environment and lifestyle. Precision medicine relies heavily on developments in systems biology and omics disciplines, including metabolomics. Combination of metabolomics with sophisticated bioinformatics analysis and mathematical modeling has an extreme power to provide a metabolic snapshot of the patient over the course of disease and treatment or classifying patients into subpopulations and subgroups requiring individual medical treatment. Although a powerful approach, metabolomics have certain limitations in technology and bioinformatics. We will review various aspects of metabolomics technology and bioinformatics, from data generation, bioinformatics analysis, data fusion and mathematical modeling to data management, in the context of precision medicine., (© The Author(s) 2018. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Squamous cell carcinoma (SCC), a malignancy arising across multiple anatomical sites, is responsible for significant cancer mortality due to insufficient therapeutic options. Here, we identify exceptional glucose reliance among SCCs dictated by hyperactive GLUT1-mediated glucose influx. Mechanistically, squamous lineage transcription factors p63 and SOX2 transactivate the intronic enhancer cluster of SLC2A1. Elevated glucose influx fuels generation of NADPH and GSH, thereby heightening the anti-oxidative capacity in SCC tumors. Systemic glucose restriction by ketogenic diet and inhibiting renal glucose reabsorption with SGLT2 inhibitor precipitate intratumoral oxidative stress and tumor growth inhibition. Furthermore, reduction of blood glucose lowers blood insulin levels, which suppresses PI3K/AKT signaling in SCC cells. Clinically, we demonstrate a robust correlation between blood glucose concentration and worse survival among SCC patients. Collectively, this study identifies the exceptional glucose reliance of SCC and suggests its candidacy as a highly vulnerable cancer type to be targeted by systemic glucose restriction., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Amino acid analysis is a powerful tool in life sciences. Current analytical methods used for the detection and quantitation of low abundance amino acids in complex samples face intrinsic challenges such as insufficient sensitivity, selectivity, and throughput. This chapter describes a protocol that makes use of AccQ•Tag chemical derivatization combined with the exceptional chromatographic resolution of ultra-performance liquid chromatography (UPLC) and the sensitivity and selectivity of tandem mass spectrometry (MS/MS). The method has been fully implemented and validated using different tandem quadrupole detectors and thoroughly tested for a variety of samples such as P. falciparum, human red blood cells, and Arabidopsis thaliana extracts. Compared to currently available methods for amino acid analysis, the AccQ•Tag UPLC-MS/MS method presented here provides enhanced sensitivity and reproducibility and offers excellent performance within a short analysis time and a broad dynamic range of analyte concentration. The focus of this chapter is the application of this improved protocol for the compositional amino acid analysis in Arabidopsis thaliana leaf extracts using the Xevo TQ for mass spectrometric detection.