Full Complement Research Articles

Protein, Amino Acids and Yield of Lentil (Lens culneris L.) Expressed Differential Response to Incremental Doses of Sulphur and Boron in Rice Fallow Soil of Mirzapur

Background: Lentil is an important legume crop and plays an important role in human nutrition and soil fertility improvement. It contains 57-60% carbohydrate, 24-26% protein, 3.2% fiberand only 1.3% fat with the full complement of amino acids. It is a potential crop for about 10 million hectares of rice-fallow areas of eastern India. This study aimed to standardized Sulphur and Boron requirements of Lentil in Eastern India. Methods: The experiment was conducted in a glass house during 2018-19 and 2019-20 in a factorial completely randomized design with three replications and 16 treatment combinations of sulphur and boron. The treatment combinations were product of 4 levels each of sulphur and boron as 0, 15, 30 and 45 kg ha-1 (0, 0.067, 0.134 and 0.201 g pot-1) and 0, 1, 2 and 3 kg ha-1 (0, 0.004, 0.009 and 0.013 g pot-1), respectively. Lentil grain yield was observed at harvest and sulphur-containing amino acids, as well as seed nutrient content, were analyzed followingstandard operating procedures. Result: Lentil grain yield was enhanced by 1.85 times with a combined application of 45 kg S and 2 kg B ha-1 with a recommended dose of NPK. The sulphur-containing amino acids namely methionine, cystine and cysteine content showed significant improvement with increasing levels of sulphur application however were not significantly affected with boron levels. The protein content in lentil seed increased considerably with application of sulphur levels as compared to boron levels and it was highest with 30kg S ha-1 + 2kg B ha-1 along with recommended dose of NPK fertilizers.

Zebrafish null mutants of Sept6 and Sept15 are viable but more susceptible to Shigella infection.

Septins are evolutionarily conserved GTP-binding proteins known for their roles in cell division and host defence against Shigella infection. Although septin group members are viewed to function as hetero-oligomeric complexes, the role of individual septins within these complexes or in isolation is poorly understood. Decades of work using mouse models has shown that some septins (including SEPT7) are essential for animal development, while others (including SEPT6) are dispensable, suggesting that some septins may compensate for the absence of others. The zebrafish genome encodes 19 septin genes, representing the full complement of septin groups described in mice and humans. In this report, we characterise null mutants for zebrafish Sept6 (a member of the SEPT6 group) and Sept15 (a member of the SEPT7 group) and test their role in zebrafish development and host defence against Shigella infection. We show that null mutants for Sept6 and Sept15 are both viable, and that expression of other zebrafish septins are not significantly affected by their mutation. Consistent with previous reports using knockdown of Sept2, Sept7b, and Sept15, we show that Sept6 and Sept15 are required for host defence against Shigella infection. These results highlight Shigella infection of zebrafish as a powerful system to study the role of individual septins in vivo.

Extracellular Vesicles as Drug Delivery Systems in Organ Transplantation: The Next Frontier.

Extracellular vesicles are lipid bilayer-delimited nanoparticles excreted into the extracellular space by all cells. They carry a cargo rich in proteins, lipids and DNA, as well as a full complement of RNA species, which they deliver to recipient cells to induce downstream signalling, and they play a key role in many physiological and pathological processes. There is evidence that native and hybrid EVs may be used as effective drug delivery systems, with their intrinsic ability to protect and deliver a functional cargo by utilising endogenous cellular mechanisms making them attractive as therapeutics. Organ transplantation is the gold standard for treatment for suitable patients with end-stage organ failure. However, significant challenges still remain in organ transplantation; prevention of graft rejection requires heavy immunosuppression and the lack of donor organs results in a failure to meet demand, as manifested by growing waiting lists. Pre-clinical studies have demonstrated the ability of EVs to prevent rejection in transplantation and mitigate ischemia reperfusion injury in several disease models. The findings of this work have made clinical translation of EVs possible, with several clinical trials actively recruiting patients. However, there is much to be uncovered, and it is essential to understand the mechanisms behind the therapeutic benefits of EVs. Machine perfusion of isolated organs provides an unparalleled platform for the investigation of EV biology and the testing of the pharmacokinetic and pharmacodynamic properties of EVs. This review classifies EVs and their biogenesis routes, and discusses the isolation and characterisation methods adopted by the international EV research community, before delving into what is known about EVs as drug delivery systems and why organ transplantation represents an ideal platform for their development as drug delivery systems.

Brain tumour segmentation with incomplete imaging data.

Progress in neuro-oncology is increasingly recognized to be obstructed by the marked heterogeneity-genetic, pathological, and clinical-of brain tumours. If the treatment susceptibilities and outcomes of individual patients differ widely, determined by the interactions of many multimodal characteristics, then large-scale, fully-inclusive, richly phenotyped data-including imaging-will be needed to predict them at the individual level. Such data can realistically be acquired only in the routine clinical stream, where its quality is inevitably degraded by the constraints of real-world clinical care. Although contemporary machine learning could theoretically provide a solution to this task, especially in the domain of imaging, its ability to cope with realistic, incomplete, low-quality data is yet to be determined. In the largest and most comprehensive study of its kind, applying state-of-the-art brain tumour segmentation models to large scale, multi-site MRI data of 1251 individuals, here we quantify the comparative fidelity of automated segmentation models drawn from MR data replicating the various levels of completeness observed in real life. We demonstrate that models trained on incomplete data can segment lesions very well, often equivalently to those trained on the full completement of images, exhibiting Dice coefficients of 0.907 (single sequence) to 0.945 (complete set) for whole tumours and 0.701 (single sequence) to 0.891 (complete set) for component tissue types. This finding opens the door both to the application of segmentation models to large-scale historical data, for the purpose of building treatment and outcome predictive models, and their application to real-world clinical care. We further ascertain that segmentation models can accurately detect enhancing tumour in the absence of contrast-enhancing imaging, quantifying the burden of enhancing tumour with an R 2 > 0.97, varying negligibly with lesion morphology. Such models can quantify enhancing tumour without the administration of intravenous contrast, inviting a revision of the notion of tumour enhancement if the same information can be extracted without contrast-enhanced imaging. Our analysis includes validation on a heterogeneous, real-world 50 patient sample of brain tumour imaging acquired over the last 15 years at our tertiary centre, demonstrating maintained accuracy even on non-isotropic MRI acquisitions, or even on complex post-operative imaging with tumour recurrence. This work substantially extends the translational opportunity for quantitative analysis to clinical situations where the full complement of sequences is not available and potentially enables the characterization of contrast-enhanced regions where contrast administration is infeasible or undesirable.

Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved metabolic enzyme that catalyzes the interconversion of isocitrate and α-ketoglutarate. Kinetic and structural studies with IDH1 have revealed evidence of striking conformational changes that occur upon binding of its substrates, isocitrate and NADP+, and its catalytic metal cation. Here, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to build a comprehensive map of the dynamic conformational changes experienced by IDH1 upon ligand binding. IDH1 proved well-suited for HDX-MS analysis, allowing us to capture profound changes in solvent accessibility at substrate binding sites and at a known regulatory region, as well as at more distant local subdomains that appear to support closure of this protein into its active conformation. HDX-MS analysis suggested that IDH1 is primarily purified with NADP(H) bound in the absence of its metal cation. Subsequent metal cation binding, even in the absence of isocitrate, was critical for driving large conformational changes. WT IDH1 folded into its fully closed conformation only when the full complement of substrates and metal was present. Finally, we show evidence supporting a previously hypothesized partially open conformation that forms prior to the catalytically active state, and we propose this conformation is driven by isocitrate binding in the absence of metal.

Nrf2 and Antioxidant Response in Animal Models of Type 2 Diabetes.

This perspective examines the proposition that chronically elevated blood glucose levels caused by type 2 diabetes (T2D) harm body tissues by locally generating reactive oxygen species (ROS). A feed-forward scenario is described in which the initial onset of defective beta cell function T2D becomes sustained and causes chronic elevations in blood glucose, which flood metabolic pathways throughout the body, giving rise to abnormally high local levels of ROS. Most cells can defend themselves via a full complement of antioxidant enzymes that are activated by ROS. However, the beta cell itself does not contain catalase or glutathione peroxidases and thereby runs a greater risk of ROS-induced damage. In this review, previously published experiments are revisited to examine the concept that chronic hyperglycemia can lead to oxidative stress in the beta cell, how this relates to the absence of beta cell glutathione peroxidase (GPx) activity, and whether this deficiency might be ameliorated by genetic enrichment of beta cell GPx and by oral antioxidants, including ebselen, a GPx mimetic.

Enantio- and Diastereoselective De Novo Synthesis of 3-Substituted Proline Derivatives via Cooperative Photoredox/Brønsted Acid Catalysis and Epimerization.

Herein, a novel strategy for the catalytic asymmetric synthesis of enantioenriched 3-cis- and 3-trans-substituted prolines has been successfully established via an unprecedented cascade radical addition/cyclization enabled by synergistic photoredox/Brønsted acid catalysis and subsequent base-assisted epimerization. The current protocol provides a unique de novo access to all four stereoisomers of 3-substituted prolines which are not readily achieved via currently established methods. This methodology could be further extended to the asymmetric synthesis of the full complement of stereoisomers of 3-substituted pipecolinic acids.

Age-related deficits in neuronal physiology and cognitive function are recapitulated in young mice overexpressing the L-type calcium channel, CaV 1.3.

The calcium dysregulation hypothesis of brain aging posits that an age-related increase in neuronal calcium concentration is responsible for alterations in a variety of cellular processes that ultimately result in learning and memory deficits in aged individuals. We previously generated a novel transgenic mouse line, in which expression of the L-type voltage-gated calcium, CaV 1.3, is increased by ~50% over wild-type littermates. Here, we show that, in young mice, this increase is sufficient to drive changes in neuronal physiology and cognitive function similar to those observed in aged animals. Specifically, there is an increase in the magnitude of the postburst afterhyperpolarization, a deficit in spatial learning and memory (assessed by the Morris water maze), a deficit in recognition memory (assessed in novel object recognition), and an overgeneralization of fear to novel contexts (assessed by contextual fear conditioning). While overexpression of CaV 1.3 recapitulated these key aspects of brain aging, it did not produce alterations in action potential firing rates, basal synaptic communication, or spine number/density. Taken together, these results suggest that increased expression of CaV 1.3 in the aged brain is a crucial factor that acts in concert with age-related changes in other processes to produce the full complement of structural, functional, and behavioral outcomes that are characteristic of aged animals.

Meiotic resetting of the cellular Sod1 pool is driven by protein aggregation, degradation, and transient LUTI-mediated repression.

Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case-Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients-is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1's role in human disease.

Use of Kidneys from Anencephalic Donors to Offset Organ Shortage

Background: It is well recognized that patient survival and quality of life are superior with renal transplantation than with dialysis. Organ availability is far outweighed by the large number of wait-listed patients. Additional stratagems are sought to expand the donor pool, and kidneys from anencephalic infants can be considered a source of organs, until now unexplored. We plan to assess the feasibility of using the kidneys from anencephalic infants for transplantation. Material and Methods: Information about anencephaly, the characteristics of the infant kidneys, the ethical, social and medico-legal aspects raised by the use of these kidneys, their procurement and their transplantation are reviewed. Conclusions: En bloc kidney transplants from infants can provide long-term normal renal function after an accelerated catch up growth. They are not subjected to hyperfiltration since they have a full complement of nephrons. They can be transplanted using the techniques currently available.

Measurement of O2 Binding by Sensory Hemeproteins.

The discovery of an increasing number of proteins that function in the detoxification and sensing of gaseous ligands has renewed interest in hemeproteins. It is critical to measure the affinities of these proteins for ligands like O2, CO, and NO, know with confidence when a protein is fully saturated with a specific ligand, and be able to estimate how well a ligand will compete against other ligands for a specific protein. Below we describe how to obtain an intact O2-binding hemeprotein with a full complement of heme, how to evaluate the factors that can impact its affinity for O2, and how to determine accurately the equilibrium and kinetic parameters Kd, kon, and koff for O2 binding.

Combined Heterozygous Genetic Variations in Complement C2 and C8B: An Explanation for Multidimensional Immune Imbalance?

The complement system plays a crucial role in host defense, homeostasis, and tissue regeneration and bridges the innate and the adaptive immune systems. Although the genetic variants in complement C2 (c.839_849+17del; p.(Met280Asnfs*5)) and C8B (c.1625C>T; p.(Thr542Ile)) are known individually, here, we report on a patient carrying their combination in a heterozygous form. The patient presented with a reduced general condition and suffers from a wide variety of autoimmune diseases. While no autoimmune disease-specific autoantibodies could be detected, genetic analysis revealed abnormalities in the two complement genes C2 and C8B. Therefore, we performed a comprehensive investigation of the innate immune system on a cellular and humoral level to define the functional consequences. We found slightly impaired functionality of neutrophils and monocytes regarding phagocytosis and reactive oxygen species generation and a diminished expression of the C5aR1. An extensive complement analysis revealed a declined activation potential for the alternative and classical pathway. Reconstitution with purified C2 and C8 into patient serum failed to normalize the dysfunction, whereas the addition of C3 improved the hemolytic activity. In clinical transfer, in vitro supplementation of the patient’s plasma with FFP as a complement source could fully restore full complement functionality. This study describes for the first time a combined heterozygous genetic variation in complement C2 and C8B which, however, cannot fully explain the overall dysfunctions and calls for further complement deficiency research and corresponding therapies.

Plant-specific features of respiratory supercomplex I + III2 from Vigna radiata

The last steps of cellular respiration—an essential metabolic process in plants—are carried out by mitochondrial oxidative phosphorylation. This process involves a chain of multi-subunit membrane protein complexes (complexes I–V) that form higher-order assemblies called supercomplexes. Although supercomplexes are the most physiologically relevant form of the oxidative phosphorylation complexes, their functions and structures remain mostly unknown. Here we present the cryogenic electron microscopy structure of the supercomplex I + III2 from Vigna radiata (mung bean). The structure contains the full subunit complement of complex I, including a newly assigned, plant-specific subunit. It also shows differences in the mitochondrial processing peptidase domain of complex III2 relative to a previously determined supercomplex with complex IV. The supercomplex interface, while reminiscent of that in other organisms, is plant specific, with a major interface involving complex III2’s mitochondrial processing peptidase domain and no participation of complex I’s bridge domain. The complex I structure suggests that the bridge domain sets the angle between the enzyme’s two arms, limiting large-scale conformational changes. Moreover, complex I’s catalytic loops and its response in active-to-deactive assays suggest that, in V. radiata, the resting complex adopts a non-canonical state and can sample deactive- or open-like conformations even in the presence of substrate. This study widens our understanding of the possible conformations and behaviour of complex I and supercomplex I + III2. Further studies of complex I and its supercomplexes in diverse organisms are needed to determine the universal and clade-specific mechanisms of respiration.

The Memphis Pandemic Health Informatics System (MEMPHI-SYS)-Creating a Metropolitan COVID-19 Data Registry Linked Directly to Community Testing to Enhance Population Health Surveillance.

The current coronavirus disease (COVID-19) pandemic has placed unprecedented strain on underfunded public health resources in the Southeastern United States. The Memphis, TN, metropolitan region has lacked infrastructure for health data exchange.This manuscript describes a multidisciplinary initiative to create a community-focused COVID-19 data registry, the Memphis Pandemic Health Informatics System (MEMPHI-SYS). MEMPHI-SYS leverages test result data updated directly from community-based testing sites, as well as a full complement of public health data sets and knowledge-based informatics. It has been guided by relationships with community stakeholders and is managed alongside the largest publicly funded community-based COVID-19 testing response in the Mid-South. MEMPHI-SYS has supported interactive Web-based analytic resources and informs federally funded COVID-19 outreach directed toward neighborhoods most in need of pandemic support.MEMPHI-SYS provides an instructive case study of how to collaboratively establish the technical scaffolding and human relationships necessary for data-driven, health equity-focused pandemic surveillance, and policy interventions.

Gene Loss may have Shaped the Cnidarian and Bilaterian Hox and ParaHox Complement.

Hox and ParaHox transcription factors are important for specifying cell fates along the primary body axes during the development of most animals. Within Cnidaria, much of the research on Hox/ParaHox genes has focused on Anthozoa (anemones and corals) and Hydrozoa (hydroids) and has concentrated on the evolution and function of cnidarian Hox genes in relation to their bilaterian counterparts. Here we analyze together the full complement of Hox and ParaHox genes from species representing all four medusozoan classes (Staurozoa, Cubozoa, Hydrozoa, and Scyphozoa) and both anthozoan classes (Octocorallia and Hexacorallia). Our results show that Hox genes involved in patterning the directive axes of anthozoan polyps are absent in the stem leading to Medusozoa. For the first time, we show spatial and temporal expression patterns of Hox and ParaHox genes in the upside-down jellyfish Cassiopea xamachana (Scyphozoa), which are consistent with diversification of medusozoan Hox genes both from anthozoans and within medusozoa. Despite unprecedented taxon sampling, our phylogenetic analyses, like previous studies, are characterized by a lack of clear homology between most cnidarian and bilaterian Hox and Hox-related genes. Unlike previous studies, we propose the hypothesis that the cnidarian-bilaterian ancestor possessed a remarkably large Hox complement and that extensive loss of Hox genes was experienced by both cnidarian and bilaterian lineages.

Opportunities for Accelerating Drug Discovery and Development by Using Engineered Drug-Metabolizing Enzymes.

The study of drug metabolism is fundamental to drug discovery and development (DDD) since by mediating the clearance of most drugs, metabolic enzymes influence their bioavailability and duration of action. Biotransformation can also produce pharmacologically active or toxic products, which complicates the evaluation of the therapeutic benefit versus liability of potential drugs but also provides opportunities to explore the chemical space around a lead. The structures and relative abundance of metabolites are determined by the substrate and reaction specificity of biotransformation enzymes and their catalytic efficiency. Preclinical drug biotransformation studies are done to quantify in vitro intrinsic clearance to estimate likely in vivo pharmacokinetic parameters, to predict an appropriate dose, and to anticipate interindividual variability in response, including from drug-drug interactions. Such studies need to be done rapidly and cheaply, but native enzymes, especially in microsomes or hepatocytes, do not always produce the full complement of metabolites seen in extrahepatic tissues or preclinical test species. Furthermore, yields of metabolites are usually limiting. Engineered recombinant enzymes can make DDD more comprehensive and systematic. Additionally, as renewable, sustainable, and scalable resources, they can also be used for elegant chemoenzymatic, synthetic approaches to optimize or synthesize candidates as well as metabolites. Here, we will explore how these new tools can be used to enhance the speed and efficiency of DDD pipelines and provide a perspective on what will be possible in the future. The focus will be on cytochrome P450 enzymes to illustrate paradigms that can be extended in due course to other drug-metabolizing enzymes. SIGNIFICANCE STATEMENT: Protein engineering can generate enhanced versions of drug-metabolizing enzymes that are more stable, better suited to industrial conditions, and have altered catalytic activities, including catalyzing non-natural reactions on structurally complex lead candidates. When applied to drugs in development, libraries of engineered cytochrome P450 enzymes can accelerate the identification of active or toxic metabolites, help elucidate structure activity relationships, and, when combined with other synthetic approaches, provide access to novel structures by regio- and stereoselective functionalization of lead compounds.

Abstract IA016: Lessons learned from clinical trials of MUC1 peptide vaccines for cancer prevention

Abstract We report on three trials of a MUC1 peptide vaccine for cancer prevention. Two trials were pilot studies evaluating vaccine immunogenicity and safety. One was in subjects with advanced colon adenomas, and the other in smokers at risk for lung cancer. The third trial was a multi-center, randomized placebo-controlled trial in subjects with advanced adenoma that included an assessment of the clinical efficacy of the vaccine to prevent recurrent adenoma. All trials successfully recruited their full complement of participants and the vaccine was well tolerated with no safety concerns. The response rate in the colon adenoma trials was 43% in the pilot study and 25% in the placebo- controlled multicenter trial. Only 10% of smokers responded to the vaccine. Higher levels of circulating myeloid derived suppressor cells (MDSC) were consistently associated with the lack of an immune response. This suggests that even in pre-malignancy immunosuppressive tendencies can impair vaccine immunogenicity. Responders to the vaccine demonstrated immune memory at one year. In the multicenter adenoma trial that evaluated the efficacy of the vaccine on adenoma recurrence, immune responders demonstrated an association with a reduced rate of adenoma recurrence. In future studies, patient selection based on circulating MDSC levels, or concomitant use of agents to counter MDSC immunosuppressive function or otherwise improve T cell function, should be considered. Citation Format: Robert E. Schoen. Lessons learned from clinical trials of MUC1 peptide vaccines for cancer prevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA016.

Investigating mangrove-human health relationships: A review of recently reported physiological benefits.

Ecosystems continue to experience degradation worldwide, with diminishing ecosystem services presenting unfavourable outlooks for all aspects of human wellbeing including health. To inform protective policies that safeguard both ecological and health benefits, syntheses of available knowledge are required especially for neglected ecosystems such as mangroves. However, reviews about relationships between mangroves and human health are rare. This review identifies and categorizes evidence reported in the Web of Science database about health impacts of mangrove ecosystem goods and services. 96 papers were retained after application of exclusion criteria and filtration steps to results of database and bibliographical searches. Findings highlight most abundantly that bioactive extracts of mangrove sediment, plant, and plant associates are useful for the treatment of human ailments and infections. Also reported is the heavy and trace metal bioremediation capacity of mangroves ecosystems, with concomitant modulating effects on associated human health risks. Evidence of mangrove influence on human nutrition via fisheries and food production support services, either singularly or in conjunction with linked ecosystems is also offered. Finally, mangrove effects on the prevalence of causative agents, and therefore on the incidence and distribution of infectious diseases, are also presented. Positive influences of mangroves on human health are implied via three of the four routes reported, which diminish with degradation and appreciate with proper ecosystem functioning. The undesirable links lie chiefly with higher infectious disease risk posed by mangroves, which requires further exploration regarding suspected ecological pathways available for limiting said risks. Other gaps identified are sparse information about in-vivo efficacy and safety of mangrove bioactive isolates, specific nutrient content and diversity associated with mangrove-supported food production outcomes, and the geographically limited nature of most findings. Beyond economic value, health benefits of mangroves are significant and outweigh their disservices to humans. To ensure sustainable supply of the full complement of these benefits, they should be considered when designing ecosystems management regimes.

A glutathione-independent DJ-1/PfpI domain-containing tomato glyoxalaseIII2, SlGLYIII2, confers enhanced tolerance under salt and osmotic stresses.

In plants, glyoxalase enzymes are activated under stress conditions to mitigate the toxic effects of hyperaccumulated methylglyoxal (MG), a highly reactive carbonyl compound. Until recently, a glutathione-dependent bi-enzymatic pathway involving glyoxalase I (GLYI) and glyoxalase II (GLYII) was considered the primary MG-detoxification system. Recently, a new glutathione-independent glyoxalase III (GLYIII) mediated direct route was also reported in plants. However, the physiological significance of this new pathway remains to be elucidated across plant species. This study identified the full complement of 22 glyoxalases in tomato. Based on their strong induction under multiple abiotic stresses, SlGLYI4, SlGLYII2 and SlGLYIII2 were selected candidates for further functional characterisation. Stress-inducible overexpression of both glutathione-dependent (SlGLYI4 + SlGLYII2) and independent (SlGLYIII2) pathways led to enhanced tolerance in both sets of transgenic plants under abiotic stresses. However, SlGLYIII2 overexpression (OE) plants outperformed the SlGLYI4 + SlGLYII2 OE counterparts for their stress tolerance under abiotic stresses. Further, knockdown of SlGLYIII2 resulted in plants with exacerbated stress responses than those silenced for both SlGLYI4 and SlGLYII2. The superior performance of SlGLYIII2 OE tomato plants for better growth and yield under salt and osmotic treatments could be attributed to better GSH/GSSG ratio, lower reactive oxygen species levels, and enhanced antioxidant potential, indicating a prominent role of GLYIII MG-detoxification pathway in abiotic stress mitigation in this species.

Unified-Hydrophilic-Interaction/Anion-Exchange Liquid Chromatography Mass Spectrometry (Unified-HILIC/AEX/MS): A Single-Run Method for Comprehensive and Simultaneous Analysis of Polar Metabolome.

One of the technical challenges in the field of metabolomics is the development of a single-run method to detect the full complement of polar metabolites in biological samples. However, an ideal method to meet this demand has not yet been developed. Herein, we proposed a simple methodology that enables the comprehensive and simultaneous analysis of polar metabolites using unified-hydrophilic-interaction/anion-exchange liquid chromatography mass spectrometry (unified-HILIC/AEX/MS) with a polymer-based mixed amines column composed of methacrylate-based polymer particles with primary, secondary, tertiary, and quaternary amines as functional groups. The optimized unified-HILIC/AEX/MS method is composed of two consecutive chromatographic separations, HILIC-dominant separation for cationic, uncharged, and zwitterionic polar metabolites [retention times (RTs) = 0-12.8 min] and AEX-dominant separation for polar anionic metabolites (RTs = 12.8-26.5 min), by varying the ratio of acetonitrile to 40 mM ammonium bicarbonate solution (pH 9.8). A total of 400 polar metabolites were analyzed simultaneously through a combination of highly efficient separation using unified-HILIC/AEX and remarkably sensitive detection using multiple reaction monitoring-based triple quadrupole mass spectrometry (unified-HILIC/AEX/MS/MS). A nontargeted metabolomic approach using unified-HILIC/AEX high-resolution mass spectrometry (unified-HILIC/AEX/HRMS) also provided more comprehensive information on polar metabolites (3242 metabolic features) in HeLa cell extracts than the conventional HILIC/HRMS method (2068 metabolic features). Our established unified-HILIC/AEX/MS/MS and unified-HILIC/AEX/HRMS methods have several advantages over conventional techniques, including polar metabolome coverage, throughput, and accurate quantitative performance, and represent potentially useful tools for in-depth studies on metabolism and biomarker discovery.