Reactive Electrophilic Species Research Articles

Singlet-Oxygen-Mediated Regulation of Photosynthesis-Specific Genes: A Role for Reactive Electrophiles in Signal Transduction.

During photosynthesis, reactive oxygen species (ROS) are formed, including hydrogen peroxide (H2O2) and singlet oxygen (1O2), which have putative roles in signalling, but their involvement in photosynthetic acclimation is unclear. Due to extreme reactivity and a short lifetime, 1O2 signalling occurs via its reaction products, such as oxidised poly-unsaturated fatty acids in thylakoid membranes. The resulting lipid peroxides decay to various aldehydes and reactive electrophile species (RES). Here, we investigated the role of ROS in the signal transduction of high light (HL), focusing on GreenCut2 genes unique to photosynthetic organisms. Using RNA seq. data, the transcriptional responses of Chlamydomonas reinhardtii to 2 h HL were compared with responses under low light to exogenous RES (acrolein; 4-hydroxynonenal), β-cyclocitral, a β-carotene oxidation product, as well as Rose Bengal, a 1O2-producing photosensitiser, and H2O2. HL induced significant (p < 0.05) up- and down-regulation of 108 and 23 GreenCut2 genes, respectively. Of all HL up-regulated genes, over half were also up-regulated by RES, including RBCS1 (ribulose bisphosphate carboxylase small subunit), NPQ-related PSBS1 and LHCSR1. Furthermore, 96% of the genes down-regulated by HL were also down-regulated by 1O2 or RES, including CAO1 (chlorophyllide-a oxygnease), MDH2 (NADP-malate dehydrogenase) and PGM4 (phosphoglycerate mutase) for glycolysis. In comparison, only 0-4% of HL-affected GreenCut2 genes were similarly affected by H2O2 or β-cyclocitral. Overall, 1O2 plays a significant role in signalling during the initial acclimation of C. reinhardtii to HL by up-regulating photo-protection and carbon assimilation and down-regulating specific primary metabolic pathways. Our data support that this pathway involves RES.

The Light-activated Effect of Natural Anthraquinone Parietin against Candida auris and Other Fungal Priority Pathogens

AbstractAntimicrobial photodynamic therapy (aPDT) is an evolving treatment strategy against human pathogenic microbes such as the Candida species, including the emerging pathogen C. auris. Using a modified EUCAST protocol, the light-enhanced antifungal activity of the natural compound parietin was explored. The photoactivity was evaluated against three separate strains of five yeasts, and its molecular mode of action was analysed via several techniques, i.e., cellular uptake, reactive electrophilic species (RES), and singlet oxygen yield. Under experimental conditions (λ = 428 nm, H = 30 J/cm2, PI = 30 min), microbial growth was inhibited by more than 90% at parietin concentrations as low as c = 0.156 mg/L (0.55 µM) for C. tropicalis and Cryptococcus neoformans, c = 0.313 mg/L (1.10 µM) for C. auris, c = 0.625 mg/L (2.20 µM) for C. glabrata, and c = 1.250 mg/L (4.40 µM) for C. albicans. Mode-of-action analysis demonstrated fungicidal activity. Parietin targets the cell membrane and induces cell death via ROS-mediated lipid peroxidation after light irradiation. In summary, parietin exhibits light-enhanced fungicidal activity against all Candida species tested (including C. auris) and Cryptococcus neoformans, covering three of the four critical threats on the WHOʼs most recent fungal priority list.

Plasmalogen oxidation induces the generation of excited molecules and electrophilic lipid species.

Plasmalogens are glycerophospholipids with a vinyl ether linkage at the sn-1 position of the glycerol backbone. Despite being suggested as antioxidants due to the high reactivity of their vinyl ether groups with reactive oxygen species, our study reveals the generation of subsequent reactive oxygen and electrophilic lipid species from oxidized plasmalogen intermediates. By conducting a comprehensive analysis of the oxidation products by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), we demonstrate that singlet molecular oxygen [O2 (1Δg)] reacts with the vinyl ether bond, producing hydroperoxyacetal as a major primary product (97%) together with minor quantities of dioxetane (3%). Furthermore, we show that these primary oxidized intermediates are capable of further generating reactive species including excited triplet carbonyls and O2 (1Δg) as well as electrophilic phospholipid and fatty aldehyde species as secondary reaction products. The generation of excited triplet carbonyls from dioxetane thermal decomposition was confirmed by light emission measurements in the visible region using dibromoanthracene as a triplet enhancer. Moreover, O2 (1Δg) generation from dioxetane and hydroperoxyacetal was evidenced by detection of near-infrared light emission at 1,270 nm and chemical trapping experiments. Additionally, we have thoroughly characterized alpha-beta unsaturated phospholipid and fatty aldehydes by LC-HRMS analysis using two probes that specifically react with aldehydes and alpha-beta unsaturated carbonyls. Overall, our findings demonstrate the generation of excited molecules and electrophilic lipid species from oxidized plasmalogen species unveiling the potential prooxidant nature of plasmalogen-oxidized products.

Genetic characterization of glyoxalase pathway in oral streptococci and its contribution to interbacterial competition

ABSTRACT Objectives To analyze contributions to microbial ecology of Reactive Electrophile Species (RES), including methylglyoxal, generated during glycolysis. Methods Genetic analyses were performed on the glyoxalase pathway in Streptococcus mutans (SM) and Streptococcus sanguinis (SS), followed by phenotypic assays and transcription analysis. Results Deleting glyoxalase I (lguL) reduced RES tolerance to a far greater extent in SM than in SS, decreasing the competitiveness of SM against SS. Although SM displays a greater RES tolerance than SS, lguL-null mutants of either species showed similar tolerance; a finding consistent with the ability of methylglyoxal to induce the expression of lguL in SM, but not in SS. A novel paralogue of lguL (named gloA2) was identified in most streptococci. SM mutant ∆gloA2SM showed little change in methylglyoxal tolerance yet a significant growth defect and increased autolysis on fructose, a phenotype reversed by the addition of glutathione, or by the deletion of a fructose: phosphotransferase system (PTS) that generates fructose-1-phosphate (F-1-P). Conclusions Fructose contributes to RES generation in a PTS-specific manner, and GloA2 may be required to degrade certain RES derived from F-1-P. This study reveals the critical roles of RES in fitness and interbacterial competition and the effects of PTS in modulating RES metabolism.

Thianthrene/TfOH-catalyzed electrophilic halogenations using N-halosuccinimides as the halogen source.

Organohalides are vital organic building blocks with applications spanning various fields. However, direct halogenation of certain neutral or unreactive substrates by using solely the regular halogenating reagents has proven challenging. Although various halogenation approaches via activating halogenating reagents or substrates have emerged, a catalytic system enabling broad substrate applicability and diverse halogenation types remains relatively underexplored. Inspired by the halogenation of arenes via thianthrenation of arenes, here we report that thianthrene, in combined use with trifluoromethanesulfonic acid (TfOH), could work as an effective catalytic system to activate regular halogenating reagents (NXS). This new protocol could accomplish multiple types of halogenation of organic compounds including aromatics, olefins, alkynes and ketones. The mechanism study indicated that a highly reactive electrophilic halogen thianthrenium species, formed in situ from the reaction of NXS with thianthrene in the presence of TfOH, was crucial for the efficient halogenation process.

Staphylococcus aureus adapts to the immunometabolite itaconic acid by inducing acid and oxidative stress responses including S-bacillithiolations and S-itaconations

Staphylococcus aureus is a major pathogen, which has to defend against reactive oxygen and electrophilic species encountered during infections. Activated macrophages produce the immunometabolite itaconate as potent electrophile and antimicrobial upon pathogen infection. In this work, we used transcriptomics, metabolomics and shotgun redox proteomics to investigate the specific stress responses, metabolic changes and redox modifications caused by sublethal concentrations of itaconic acid in S. aureus.In the RNA-seq transcriptome, itaconic acid caused the induction of the GlnR, KdpDE, CidR, SigB, GraRS, PerR, CtsR and HrcA regulons and the urease-encoding operon, revealing an acid and oxidative stress response and impaired proteostasis. Neutralization using external urea as ammonium source improved the growth and decreased the expression of the glutamine synthetase-controlling GlnR regulon, indicating that S. aureus experienced ammonium starvation upon itaconic acid stress. In the extracellular metabolome, the amounts of acetate and formate were decreased, while secretion of pyruvate and the neutral product acetoin were strongly enhanced to avoid intracellular acidification. Exposure to itaconic acid affected the amino acid uptake and metabolism as revealed by the strong intracellular accumulation of lysine, threonine, histidine, aspartate, alanine, valine, leucine, isoleucine, cysteine and methionine. In the proteome, itaconic acid caused widespread S-bacillithiolation and S-itaconation of redox-sensitive antioxidant and metabolic enzymes, ribosomal proteins and translation factors in S. aureus, supporting its oxidative and electrophilic mode of action in S. aureus. In phenotype analyses, the catalase KatA, the low molecular weight thiol bacillithiol and the urease provided protection against itaconic acid-induced oxidative and acid stress in S. aureus. Altogether, our results revealed that under physiological infection conditions, such as in the acidic phagolysome, itaconic acid is a highly effective antimicrobial against multi-resistant S. aureus isolates, which acts as weak acid causing an acid, oxidative and electrophilic stress response, leading to S-bacillithiolation and itaconation.

Structural Insights into Electrophiles and Electrophilic Metabolites Sensing and Signaling: The Missing Information

AbstractNative reactive electrophilic species (RES) are long‐recognized regulators of pathophysiology; yet, knowledge surrounding how RES regulate context‐specific biology remains limited. The latest technological advances in profiling and precision decoding of RES sensing and signaling have begun to bring about improved understanding of localized RES regulatory paradigms. However, studies in purified systems — prerequisites for gaining structure/function insights — prove challenging. We here introduce emerging chemical biology tools available to probe RES signaling, and the new knowledge that these tools have brought to the field. We next discuss existing structural data of RES‐sensor proteins complexed with electrophilic metabolites or small molecule drugs (limited to &lt;300 Da), including challenges faced in acquiring homogenous RES‐bound proteins. We further offer considerations that could promote enhanced understanding of RES regulation derived from three‐dimensional structures of RES‐modified proteins.

Cysteine thiol-based post-translational modification: What do we know about transcription factors?

Reactive electrophilic species are ubiquitous in plant cells, where they contribute to specific redox-regulated signaling events. Redox signaling is known to modulate gene expression during diverse biological processes, including plant growth, development, and environmental stress responses. Emerging data demonstrates that transcription factors (TFs) are a main target of cysteine thiol-based oxidative post-translational modifications (OxiPTMs), which can alter their transcriptional activity and thereby convey redox information to the nucleus. Here, we review the significant progress that has been made in characterizing cysteine thiol-based OxiPTMs, their biochemical properties, and their functional effects on plant TFs. We discuss the underlying mechanism of redox regulation and its contribution to various physiological processes as well as still outstanding challenges in redox regulation of plant gene expression.

Olive leaf (Olea europaea L. folium) extract influences liver microsomal detoxifying enzymes in rats orally exposed to 2-amino-l-methyI-6-phenyI-imidazo pyridine (PhIP).

Olive tree (Olea europaea, Oleaceae) leaf extract (OLE) exerts many biological activities. One of the most common polycyclic aromatic hydrocarbons (PAHs) that pollute the environment is 2-amino-l-methyI-6-phenyI-imidazo pyridine (PhIP). It is a food-derived carcinogen that is present in fish and meat that has been cooked at high temperatures. Due to the generation of reactive electrophilic species, phase I enzymes have the potential to cause oxidative damage. In order to safely remove these reactive species from the body, phase II detoxification (conjugation) enzymes are necessary. It is not known whether OLE could influence their activities and hence reduce the carcinogenic effects of PhIP. This study evaluated whether OLE could modulate phase I detoxifying enzymes as well as phase II enzymes that metabolize PhIP in rat liver microsomes. Four groups of rats were used: group I: no treatment; group II: OLE (10mg/kg bw orally); group III: PhIP (0.1mg/kg bw orally); and group IV: PhIP followed by OLE. After 4weeks, the activities of phase I enzymes such as CYP1A1 (ethoxyresorufin O-deethylase), CYP2E1 (p-nitrophenol hydroxylase), CYP1A2 (methoxyresorufin O-demethylase), UDP-glucuronyl transferase, sulphotransferase, and glutathione-S transferase were evaluated in rat liver microsomes. Analysis of OLE by gas chromatography-mass spectrometry (GC/MS) showed various active ingredients in OLE, including 3,5-Heptadienal (C10H14O), 3,4-dimethoxy benzoic acid (C8H10O3), 4-hydroxy-3-methoxy (C8H8O4), 1,3,5-Benzenetriol (C6H6O3), hexadecanoic acid (C16H32O2), and hexadecanoic acid ethyl ester (C18H36O2). Our results showed that rats given PhIP were found to have a statistically significant (p < 0.001) reduction in the activities of CYP1A1, CYP1A2, and CYP2E1 in comparison with the control group. However, treatment with OLE enhanced their activities but not to a normal level compared with untreated groups. Administration of PhIP decreased the activities of phase II enzymes (glutathione S-transferase, UDP-glucuronyltransferase, or sulphotransferase) (p < 0.01) in comparison with the control group. Histological examination of rat livers was consistent with the biochemical changes. The administration of OLE improved the phase II enzyme activities in animals injected with PhIP. We conclude that OLE influences phase I and phase II detoxification enzymes exposed to PhIP, which may represent a new approach to attenuating carcinogenesis induced by it.

ROS-derived lipid peroxidation is prevented in barley leaves during senescence.

Senescence in plants enables resource recycling from senescent leaves to sink organs. Under stress, increased production of reactive oxygen species (ROS) and associated signalling activates senescence. However, senescence is not always associated with stress since it has a prominent role in plant development, in which the role of ROS signalling is less clear. To address this, we investigated lipid metabolism and patterns of lipid peroxidation related to signalling during sequential senescence in first‐emerging barley leaves grown under natural light conditions. Leaf fatty acid compositions were dominated by linolenic acid (75% of total), the major polyunsaturated fatty acid (PUFA) in galactolipids of thylakoid membranes, known to be highly sensitive to peroxidation. Lipid catabolism during senescence, including increased lipoxygenase activity, led to decreased levels of PUFA and increased levels of short‐chain saturated fatty acids. When normalised to leaf area, only concentrations of hexanal, a product from the 13‐lipoxygenase pathway, increased early upon senescence, whereas reactive electrophile species (RES) from ROS‐associated lipid peroxidation, such as 4‐hydroxynonenal, 4‐hydroxyhexenal and acrolein, as well as β‐cyclocitral derived from oxidation of β‐carotene, decreased. However, relative to total chlorophyll, amounts of most RES increased at late‐senescence stages, alongside increased levels of α‐tocopherol, zeaxanthin and non‐photochemical quenching, an energy dissipative pathway that prevents ROS production. Overall, our results indicate that lipid peroxidation derived from enzymatic oxidation occurs early during senescence in first barley leaves, while ROS‐derived lipid peroxidation associates weaker with senescence.

Reactive metabolic byproducts contribute to antibiotic lethality under anaerobic conditions

Understanding how bactericidal antibiotics kill bacteria remains an open question. Previous work has proposed that primary drug-target corruption leads to increased energetic demands, resulting in the generation of reactive metabolic byproducts (RMBs), particularly reactive oxygen species, that contribute to antibiotic-induced cell death. Studies have challenged this hypothesis by pointing to antibiotic lethality under anaerobic conditions. Here, we show that treatment of Escherichia coli with bactericidal antibiotics under anaerobic conditions leads to changes in the intracellular concentrations of central carbon metabolites, as well as the production of RMBs, particularly reactive electrophilic species (RES). We show that antibiotic treatment results in DNA double-strand breaks and membrane damage and demonstrate that antibiotic lethality under anaerobic conditions can be decreased by RMB scavengers, which reduce RES accumulation and mitigate associated macromolecular damage. This work indicates that RMBs, generated in response to antibiotic-induced energetic demands, contribute in part to antibiotic lethality under anaerobic conditions.

Synthesis of 3-Aryl-3-(Furan-2-yl)Propanoic Acid Derivatives, and Study of Their Antimicrobial Activity.

Reactions of 3-(furan-2-yl)propenoic acids and their esters with arenes in Brønsted superacid TfOH affords products of hydroarylation of the carbon–carbon double bond, 3-aryl-3-(furan-2-yl)propenoic acid derivatives. According to NMR and DFT studies, the corresponding O,C-diprotonated forms of the starting furan acids and esters should be reactive electrophilic species in these transformations. Starting compounds and their hydroarylation products, at a concentration of 64 µg/mL, demonstrate good antimicrobial activity against yeast-like fungi Candida albicans. Apart from that, these compounds suppress Escherichia coli and Staphylococcus aureus.

The Old Yellow Enzyme OfrA Fosters Staphylococcus aureus Survival via Affecting Thiol-Dependent Redox Homeostasis.

Old yellow enzymes (OYEs) are widely found in the bacterial, fungal, and plant kingdoms but absent in humans and have been used as biocatalysts for decades. However, OYEs’ physiological function in bacterial stress response and infection situations remained enigmatic. As a pathogen, the Gram-positive bacterium Staphylococcus aureus adapts to numerous stress conditions during pathogenesis. Here, we show that in S. aureus genome, two paralogous genes (ofrA and ofrB) encode for two OYEs. We conducted a bioinformatic analysis and found that ofrA is conserved among all publicly available representative staphylococcal genomes and some Firmicutes. Expression of ofrA is induced by electrophilic, oxidative, and hypochlorite stress in S. aureus. Furthermore, ofrA contributes to S. aureus survival against reactive electrophilic, oxygen, and chlorine species (RES, ROS, and RCS) via thiol-dependent redox homeostasis. At the host–pathogen interface, S. aureusΔofrA has defective survival in macrophages and whole human blood and decreased staphyloxanthin production. Overall, our results shed the light onto a novel stress response strategy in the important human pathogen S. aureus.

Glycosyl Vinylogous Carbonates as Glycosyl Donors by Metal-Free Activation.

Synthesis of glycoconjugates employs a glycosylation reaction wherein an electrophile and a nucleophile known as a glycosyl donor and an aglycon, respectively, are involved. Glycosyl donors often contain a leaving group at the anomeric carbon that upon reaction with activator(s) results in a highly reactive electrophilic species reported as an oxycarbenium ion contact pair that will then be attacked by the aglycon. Therefore, identification of the correct glycosyl donor and activation protocol is essential for the synthesis of all glycoconjugates. Recently identified [Au]/[Ag]-catalyzed activation of ethynylcyclohexyl glycosyl carbonates is one such versatile method for the synthesis of glycosides, oligosaccharides, and glycoconjugates. In this work, stable glycosyl vinylogous carbonates were identified to undergo glycosidation in the presence of a sub-stoichiometric quantity of TfOH. The reaction is fast and suitable for donors containing both C2-ethers and C2-esters. Donors positioned with C2-ethers resulted in anomeric mixtures with greater selectivity toward 1,2-cis glycosides, whereas those with C2-esters gave 1,2-trans selective glycosides. The versatility of the method is demonstrated by conducting the glycosylation with more than 25 substrates. Furthermore, the utility of the glycosyl vinylogous carbonate donors is demonstrated with the successful synthesis of the branched pentaarabinofuranoside moiety of the Mycobacterium tuberculosis cell wall.

Direct and Sequential Bioactivation of Pemigatinib to Reactive Iminium Ion Intermediates Culminates in Mechanism-Based Inactivation of Cytochrome P450 3A.

We recently established the mechanism-based inactivation (MBI) of cytochrome P450 3A (CYP3A) by the fibroblast growth factor receptor (FGFR) inhibitors erdafitinib and infigratinib. Serendipitously, our preliminary data have also revealed that pemigatinib (PEM), another clinically approved FGFR1-3 inhibitor, similarly elicited time-dependent inhibition of CYP3A. This was rather unexpected, as it was previously purported that PEM did not pose any metabolism-dependent liabilities due to the absence of glutathione-related conjugates in metabolic profiling experiments conducted in human liver microsomes. Here, we confirmed that PEM inhibited both CYP3A isoforms in a time-, concentration-, and cofactor-dependent manner consistent with MBI, with inactivator concentration at half-maximum rate constant, maximum inactivation rate constant, and partition ratio of 8.69 and 11.95 μM, 0.108 and 0.042 min-1, and approximately 44 and approximately 47 for CYP3A4 and CYP3A5, respectively. Although the rate of inactivation was diminished by coincubation with an alternative substrate or direct inhibitor of CYP3A, the inclusion of nucleophilic trapping agents afforded no such protection. Furthermore, the lack of catalytic activity recovery following dialysis and oxidation with potassium ferricyanide coupled with the absence of a spectrally resolvable peak in the Soret region collectively implied that the underlying mechanism of inactivation was not elicited via the formation of pseudo-irreversible metabolite-intermediate complexes. Finally, utilizing cyanide trapping and high-resolution mass spectrometry, we illuminated the direct and sequential oxidative bioactivation of PEM and its major O-desmethylated metabolite at its distal morpholine moiety to reactive iminium ion hard electrophilic species that could covalently inactivate CYP3A via MBI. SIGNIFICANCE STATEMENT: This study reports for the first time the covalent MBI of CYP3A by PEM and deciphered its bioactivation pathway involving the metabolic activation of PEM and its major O-desmethylated metabolite to reactive iminium ion intermediates. Following which, a unique covalent docking methodology was harnessed to unravel the structural and molecular determinants underpinning its inactivation. Findings from this study lay the foundation for future investigation of clinically relevant drug-drug interactions between PEM and concomitant substrates of CYP3A.

Postharvest Fumigation of (E)-2-Hexenal on Kiwifruit (Actinidia chinensis cv. ‘Haegeum’) Enhances Resistance to Botrytis cinerea

(E)-2-Hexenal is a reactive electrophile species released by green plants upon herbivory or pathogenic infection and induces various defense responses. In this study, (E)-2-hexenal was used as a natural alternative to artificial fumigants to prevent postharvest pathogenic infection of the ‘Haegeum’ kiwifruit (Actinidia chinensis). Integrated transcriptome-metabolome analyses were performed to investigate its effect on the induction of plant defense responses. When kiwifruit was fumigated with 100 μmol L-1 (E)-2-hexenal for 24 h at 20 °C and then inoculated with fungal pathogen Botrytis cinerea, the disease severity, as assessed by necrotic lesion size and B. cinerea biomass, was reduced compared to the control, distilled water-treated fruit. (E)-2-Hexenal treatment also led to upregulated expression of genes encoding pattern recognition receptors, signal transducer proteins, and pathogenesis-related proteins, including chitinase. Furthermore, treatment led to increased jasmonic acid and flavonoid biosynthesis. (E)-2-Hexenal fumigation therefore enhanced the resistance of kiwifruit to B. cinerea infection, presumably by facilitating the signaling network from pathogen recognition to defense gene expression and enabling the accumulation of secondary metabolites, such as flavonoids. The present study not only suggests an optimal concentration of (E)-2-hexenal fumigation for controlling postharvest disease of kiwifruit, but also broadens our understanding of the underlying molecular mechanism by which (E)-2-hexenal enhances plant resistance.

Synthesis of 3-thiocyanated chromones via TCCA/NH4SCN-mediated cyclization/thiocyanation of alkynyl aryl ketones

3-Thiocyanated chromones was conveniently synthesized from alkynyl aryl ketones using commercially available, inexpensive trichloroisocyanuric acid (TCCA) as oxidant and NH4SCN as thiocyanato (SCN) source. This metal-free approach is postulated to first in situ generate thiocyanogen chloride (Cl-SCN) from the reaction of TCCA and NH4SCN, followed by a rare efficient electrophilic thiocyano oxyfunctionalization of alkynes enabled by the reactive electrophilic species generated thereof.

RNAi-mediated gene knockdown of progesterone 5\u03b2-reductases in Digitalis lanata reduces 5\u03b2-cardenolide content

Key messageStudying RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes) in 5β-cardenolide formation.Progesterone 5β-reductases (P5βR) are assumed to catalyze the reduction of progesterone to 5β-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5β-cardenolides. P5βRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5βRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5β-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5βR1 (AY585867.1) and DlP5βR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5β-cardenolide formation, we here established several RNAi-mediated DlP5βR1 and DlP5βR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5βR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5βR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5βR2-RNAi lines but not in Dl P5βR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5βR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).

Mitohormesis reprogrammes macrophage metabolism to enforce tolerance.

Macrophages generate mitochondrial reactive oxygen and electrophilic species (mtROS, mtRES) as antimicrobials during Toll-like receptor (TLR)-dependent inflammatory responses. Whether mitochondrial stress caused by these molecules impacts macrophage function is unknown. Here, we demonstrate that both pharmacologically- and lipopolysaccharide (LPS)-driven mitochondrial stress in macrophages triggers a stress response called mitohormesis. LPS-driven mitohormetic stress adaptations occur as macrophages transition from an LPS-responsive to LPS-tolerant state where stimulus-induced proinflammatory gene transcription is impaired, suggesting tolerance is a product of mitohormesis. Indeed, like LPS, hydroxyestrogen-triggered mitohormesis suppresses mitochondrial oxidative metabolism and acetyl-CoA production needed for histone acetylation and proinflammatory gene transcription, and is sufficient to enforce an LPS-tolerant state. Thus, mtROS and mtRES are TLR-dependent signaling molecules that trigger mitohormesis as a negative feedback mechanism to restrain inflammation via tolerance. Moreover, bypassing TLR signaling and pharmacologically triggering mitohormesis represents a novel anti-inflammatory strategy that co-opts this stress response to impair epigenetic support of proinflammatory gene transcription by mitochondria.

Evaluation of Pipemidic Acid Derivatives for Potential Antimicrobial Activity Application: In silico Studies on Bioactivity

Background: Pipemidic acid is a broad-spectrum quinolone antibacterial agent for the treatment of chronic urinary tract infections against both gram-positive and gram-negative bacteria. Both quinolone and fluoroquinolone antibiotics have been useful in combating bacterial infections. However, patients suffer severe side effects when they stop taking the medication. The piperazinyl region of pipemidic acid is highly responsible for the side effects. Objective: The objective of this study is to design new compounds in which the piperazinyl region is masked by way of conjugation to benzoic acid derivatives. Methods: In silico studies were conducted using AutoDockTools software for ligand-protein docking. The docking scores were compared to the parent pipemidic acid docked to Bacterial DNA (deoxyribonucleic acid) gyrase and GABA (gamma-Aminobutyric acid) receptor from the PDB (Protein Data Bank) database. Sites of metabolism, biological activity, quantum chemical descriptors, and ADME (absorption, distribution, metabolism, and excretion) property predictions for each designed ligand were also evaluated. Results: The docking studies and biological activity predictions showed good anti-infective properties (ligand PAR03) whilst also suggesting a reduction in GABA receptor agonist activity. The performance of PAR03 correlates with its electronic properties showing electrophilic character (can generate Reactive Electrophilic Species (RES)). Conclusion: The results from this study indicate that modification of the piperazinyl region of pipemidic acid can be an effective way to improve the drug potency whilst also ensuring reduction of the associated side effects.