Insecticidal potential and risk assessment of diamide pesticides against Spodoptera frugiperda in maize crops.

The effectiveness, tolerance, and safety of pesticides must be established before their scientific or rational. This study evaluates the field control efficacy of broflanilide, tetraniliprole, and chlorantraniliprole in combating Spodoptera frugiperda in maize crops, as well as the resistance of S. frugiperda to these three diamide pesticides after exposure. By assessing field control efficiency, toxicity, effects on development and reproduction, and detoxification enzyme activity of these diamide pesticides on S. frugiperda, highlights broflanilide's significant insecticidal potential. A highly sensitive and efficient method using QuEChERS/HPLCMS/MS was developed to simultaneously detect residues of these three pesticides on maize. Initial concentrations of broflanilide, tetraniliprole, and chlorantraniliprole ranged from 2.13 to 4.02 mg/kg, with their respective half-lives varying between 1.23 and 1.51 days. Following foliar application, by the time of harvest, the terminal residue concentrations of these pesticides were all under 0.01 mg/kg. Chronic dietary intake risk assessments and cumulative chronic dietary exposure for three pesticides indicated that the general population's terminal residue concentration was within acceptable limits. Not only does this research provide valuable insights into field control efficiency, insecticidal effects, resistance, residues, and risk assessment results of broflanilide, tetraniliprole, and chlorantraniliprole on maize, but additionally, it also paves the way for setting suitable Maximum Residue Limits (MRLs) values based on pre-harvest interval values, rational dosage, and application frequency.

Oxidative stress elicits the remodeling of vimentin filaments into biomolecular condensates.

The intermediate filament protein vimentin performs an essential role in cytoskeletal interplay and dynamics, mechanosensing and cellular stress responses. In pathology, vimentin is a key player in tumorigenesis, fibrosis and infection. Vimentin filaments undergo distinct and versatile reorganizations, and behave as redox sensors. The vimentin monomer possesses a central α-helical rod domain flanked by N- and C-terminal low complexity domains. Interactions between this type of domains play an important function in the formation of phase-separated biomolecular condensates, which in turn are critical for the organization of cellular components. Here we show that several oxidants, including hydrogen peroxide and diamide, elicit the remodeling of vimentin filaments into small particles. Oxidative stress elicited by diamide induces a fast dissociation of filaments into circular, motile dots, which requires the presence of the single vimentin cysteine residue, C328. This effect is reversible, and filament reassembly can occur within minutes of oxidant removal. Diamide-elicited vimentin droplets recover fluorescence after photobleaching. Moreover, fusion of cells expressing differentially tagged vimentin allows the detection of dots positive for both tags, indicating that vimentin dots merge upon cell fusion. The aliphatic alcohol 1,6-hexanediol, known to alter interactions between low complexity domains, readily dissolves diamide-elicited vimentin dots at low concentrations, in a C328 dependent manner, and hampers reassembly. Taken together, these results indicate that vimentin oxidation promotes a fast and reversible filament remodeling into biomolecular condensate-like structures, and provide primary evidence of its regulated phase separation. Moreover, we hypothesize that filament to droplet transition could play a protective role against irreversible damage of the vimentin network by oxidative stress.

Novel Meta-Diamide Compounds Containing Sulfide Derivatives Were Designed and Synthesized as Potential Pesticides.

The meta-diamide (m-diamide) insecticide, Broflanilide, was characterized by its high efficiency, low toxicity and lack of cross-resistance with traditional GABA receptors. In accordance with the principles of drug molecular design, easily derivable sulfur with diverse bioactivities was introduced while leading with the parent Broflanilide. Twelve novel m-diamide target compounds containing sulfide derivatives were synthesized through exploration guided by the literature. Their structures were confirmed by melting points, 1H NMR, 13C NMR and HRMS. Insecticidal activity assessments revealed that most target compounds A-D exhibited 100% lethality against Plutella xylostella (P. xylostella) and Aphis craccivora Koch (A. craccivora) at 500 mg·L-1. Notably, for P. xylostella, compounds C-2, C-3, C-4 and D-2 demonstrated 60.00-100.00% insecticidal activity even at a concentration as low as 0.625 mg·L-1. As determined by structure-activity relationship (SAR) analysis, compounds with R1 = CH3 and R2 = Br (B-1, C-2 and D-2) and sulfoxide compound C-3 contained 100.00% lethality against A. craccivora at 500 mg·L-1, surpassing the lethality when leading with the parent Broflanilide in terms of efficacy. Consequently, it can be inferred that the sulfoxide compound (C-3) requires further investigation as a potential active molecule for new insecticides. These explorations provide valuable references for future research on the synthesis and insecticidal activities of sulfide-containing m-diamide compounds.

Redox processes are major regulators of leukotriene synthesis in neutrophils exposed to bacteria Salmonella typhimurium; the way to manipulate neutrophil swarming.

Neutrophils play a primary role in protecting our body from pathogens. When confronted with invading bacteria, neutrophils begin to produce leukotriene B4, a potent chemoattractant that, in cooperation with the primary bacterial chemoattractant fMLP, stimulates the formation of swarms of neutrophils surrounding pathogens. Here we describe a complex redox regulation that either stimulates or inhibits fMLP-induced leukotriene synthesis in an experimental model of neutrophils interacting with Salmonella typhimurium. The scavenging of mitochondrial reactive oxygen species by mitochondria-targeted antioxidants MitoQ and SkQ1, as well as inhibition of their production by mitochondrial inhibitors, inhibit the synthesis of leukotrienes regardless of the cessation of oxidative phosphorylation. On the contrary, antioxidants N-acetylcysteine and sodium hydrosulfide promoting reductive shift in the reversible thiol-disulfide system stimulate the synthesis of leukotrienes. Diamide that oxidizes glutathione at high concentrations inhibits leukotriene synthesis, and the glutathione precursor S-adenosyl-L-methionine prevents this inhibition. Diamide-dependent inhibition is also prevented by diphenyleneiodonium, presumably through inhibition of NADPH oxidase and NADPH accumulation. Thus, during bacterial infection, maintaining the reduced state of glutathione in neutrophils plays a decisive role in the synthesis of leukotriene B4. Suppression of excess leukotriene synthesis is an effective strategy for treating various inflammatory pathologies. Our data suggest that the use of mitochondria-targeted antioxidants may be promising for this purpose, whereas known thiol-based antioxidants, such as N-acetylcysteine, may dangerously stimulate leukotriene synthesis by neutrophils during severe pathogenic infection.

Optimization of Pharmacokinetic and In Vitro Safety Profile of a Series of Pyridine Diamide Indirect AMPK Activators.

A set of focused analogues have been generated around a lead indirect adenosine monophosphate-activated kinase (AMPK) activator to improve the rat clearance of the molecule. Analogues were focused on inhibiting amide hydrolysis by the strategic placement of substituents that increased the steric environment about the secondary amide bond between 4-aminopiperidine and pyridine-5-carboxylic acid. It was found that placing substituents at position 3 of the piperidine ring and position 4 of the pyridine could all improve clearance without significantly impacting on-target potency. Notably, trans-3-fluoropiperidine 32 reduced rat clearance from above liver blood flow to 19 mL/min/kg and improved the hERG profile by attenuating the basicity of the piperidine moiety. Oral dosing of 32 activated AMPK in mouse liver and after 2 weeks of dosing improved glucose handling in a db/db mouse model of Type II diabetes as well as lowering fasted glucose and insulin levels.

Changes of the Protein CoAlation Pattern in Response to Oxidative Stress and Capacitation in Human Spermatozoa.

The spermatozoa have limited antioxidant defences, a high polyunsaturated fatty acids content and the impossibility of synthesizing proteins, thus being susceptible to oxidative stress. High levels of reactive oxygen species (ROS) harm human spermatozoa, promoting oxidative damage to sperm lipids, proteins and DNA, leading to infertility. Coenzyme A (CoA) is a key metabolic integrator in all living cells. Recently, CoA was shown to function as a major cellular antioxidant mediated by a covalent modification of surface-exposed cysteines by CoA (protein CoAlation) under oxidative or metabolic stresses. Here, the profile of protein CoAlation was examined in sperm capacitation and in human spermatozoa treated with different oxidizing agents (hydrogen peroxide, (H2O2), diamide and tert-butyl hydroperoxide (t-BHP). Sperm viability and motility were also investigated. We found that H2O2 and diamide produced the highest levels of protein CoAlation and the greatest reduction of sperm motility without impairing viability. Protein CoAlation levels are regulated by 2-Cys peroxiredoxins (PRDXs). Capacitated spermatozoa showed lower levels of protein CoAlation than non-capacitation cells. This study is the first to demonstrate that PRDXs regulate protein CoAlation, which is part of the antioxidant response of human spermatozoa and participates in the redox regulation associated with sperm capacitation.

The ryanodine receptor mutation I4728M confers moderate-level resistance to diamide insecticides in Spodoptera litura.

BACKGROUND: The common cutworm, Spodoptera litura (Fabricius), is one of the most widespread and destructive polyphagous pests in tropical and subtropical Asia. S. litura has evolved resistance to different insecticides, including diamide insecticides. Here, we identified a ryanodine receptor (RyR) mutation (I4728M) associated with target site resistance to diamides in a field-collected population of S. litura. The contribution of this mutation to diamide resistance was investigated through establishing a near-isogenic resistant strain of S. litura. RESULTS: The ND21 population of S. litura, collected from Ningde, Fujian province of China in 2021, exhibited 130.6-fold resistance to chlorantraniliprole compared to the susceptible NJ-S strain. S. litura RyR mutation I4728M, corresponding to Plutella xylostella RyR I4790M, was identified in the ND21 population. SlRyR I4728M mutation of ND21 was introgressed into a susceptible background strain (NJ-S) with marker-assisted backcrossing. The introgressed strain named ND21-R, which was homozygous for the mutant 4728M allele, shared about 94% of the genetic background with the NJ-S strain. ND21-R strain showed moderate levels of resistance to two anthranilic diamides (19.1-fold to chlorantraniliprole, 19.7-fold to cyantraniliprole) and the phthalic diamide flubendiamide (23.4-fold). Genetic analysis showed that chlorantraniliprole resistance was autosomal, incompletely recessive and tightly linked with SlRyR I4728M mutation in the introgressed ND21-R strain of S. litura. CONCLUSION: Identification of the I4728M mutation and its contribution to diamide resistance in S. litura will help develop allelic discrimination assays for resistance monitoring and guide resistance management practices for diamides in S. litura. © 2023 Society of Chemical Industry.

Design, Synthesis, Fungicidal and Insecticidal Activities of Novel Diamide Compounds Combining Pyrazolyl and Polyfluoro-Substituted Phenyl into Alanine or 2-Aminobutyric Acid Skeletons.

Thirty novel diamide compounds combining pyrazolyl and polyfluoro-substituted phenyl groups into alanine or 2-aminobutyric acid skeletons were designed and synthesized with pyflubumide as the lead compound to develop potent and environmentally friendly pesticides. The preliminary bioassay results indicated that the new compounds containing the para-hexa/heptafluoroisopropylphenyl moiety exhibit fungicidal, insecticidal, and acaricidal activities. This is the first time that the para-hexa/heptafluoroisopropylphenyl group is a key fragment of the fungicidal activity of new N-phenyl amide compounds. Most of the target compounds exhibited moderate to good insecticidal activity against Aphis craccivora at a concentration of 400 µg/mL, and some showed moderate activity at a concentration of 200 µg/mL; in particular, compounds I-4, II-a-10, and III-26 displayed higher than 78% lethal rates at 200 µg/mL. Compound II-a-14 exhibited a 61.1% inhibition at 200 µg/mL for Tetranychus cinnabarinus. In addition, some of the target compounds exhibited good insecticidal activities against Plutella xylostella at a concentration of 200 µg/mL; the mortalities of compounds I-1, and II-a-15 were 76.7% and 70.0%, respectively. Preliminary analysis of the structure-activity relationship (SAR) indicated that the insecticidal and acaricidal activities varied significantly depending on the type of substituent and substitution pattern. The fungicidal activity results showed that compounds I-1, II-a-10, II-a-17, and III-26 exhibited good antifungal effects. Enzymatic activity experiments and in vivo efficacy of compound II-a-10 were conducted and discussed.

Inhibition of X52 Corrosion in CO2-Saturated Brine by a Dialkyl-Diamide from Coffee Bagasse Oil.

This work reports the performance of a green corrosion inhibitor with double hydrocarbon chain. The evaluated inhibitor was a dialkyl-diamide from coffee bagasse oil and its electrochemical behavior was evaluated on an API-X52 steel in CO2-saturated brine at 60 °C. The electrochemical behavior was determined by measurements of open circuit potential, polarization resistance, and electrochemical impedance spectroscopy. In addition, the thermodynamic parameters of the corrosion process were obtained in the temperature range from 40 °C to 80 °C. Electrochemical studies showed that the inhibitor is capable of suppressing metal dissolution by up to 99% at 25 ppm. On the other hand, the thermodynamic parameters indicate that when adding the inhibitor, there is a strong increase in both Ea and ΔH° values, and that as time increases, they decrease until reaching similar values to those observed in the absence of the inhibitor. Furthermore, ΔS° values tend to become more negative with immersion time because of the formation of a stable film on the metal surface.

Determination of Cyclaniliprole in Fruits and Vegetables Using Disposable Pipette Extraction Cleanup and Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Despite an outstanding agent for control of Lepidoptera, the diamide insecticide cyclaniliprole (CYCP) is a suspected carcinogen. In the present study, an analytical method was developed for the determination of CYCP in six fruits and vegetables (apple, grape, peach, bell pepper, lettuce, and tomato) using ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry. Sample preparation was carried out by the acetonitrile-salting-out extraction followed by simple and fast cleanup of disposable pipette extraction tip containing styrene divinyl benzene and/or graphitized carbon black. Satisfactory linearity (r > 0.99) was obtained in the calibration range of 0.001−1 µg mL−1. Matrix effects decreased from −9.9−−17.9% to −1.0−−7.6% after the cleanup. The recoveries of CYCP at three spike levels (0.01, 0.1, and 1 mg kg−1) from different matrices were between 75.7% and 111.5%, with the intra-day (n = 5) and inter-day (n = 15) relative standard deviations lower than 12.1%. The limit of quantification was 0.01 mg kg−1. The developed method provides a good reference for routine monitoring of CYCP in these fruits and vegetables.

Erythrocyte mitogen-activated protein kinases mediate hemolytic events under osmotic and oxidative stress and in hemolytic diseases.

p38 MAPKs are key regulators of cellular adaptation to various stress stimuli, however, their role in mediating erythrocyte cell death and hemolysis is largely unknown. We hypothesized that activation of erythrocyte p38 MAPK is a common event in the stimulation of hemolysis, and that inhibition of p38 MAPK pathways could mitigate hemolysis in hemoglobinopathies. We exposed human erythrocytes to diamide-induced oxidative stress or to hypoosmotic shock in the presence or absence of p38 MAPK inhibitors (SCIO469, SB203580, CMPD1) and used immunoblotting to determine MAPK activity and to identify possible downstream effectors of p38 MAPK. We also evaluated the impact of p38 MAPK inhibitors on stress-induced hemolysis or hypoxia-induced sickling in erythrocytes from mouse models of sickle cell disease. We found that human erythrocytes express conventional MAPKs (MKK3, p38 MAPK, MAPKAPK2) and identified differential MAPK activation pathways in each stress condition. Specifically, p38 MAPK inhibition in diamide-treated erythrocytes was associated with decreased phosphorylation of Src tyrosine kinases and Band 3 protein. Conversely, hypoosmotic shock induced MAPKAPK2 and RSK2 phosphorylation, which was inhibited by SCIO469 or CMPD1. Relevant to hemoglobinopathies, sickle cell disease was associated with increased erythrocyte MKK3, p38 MAPK, and MAPKAPK2 expression and phosphorylation as compared with erythrocytes from healthy individuals. Furthermore, p38 MAPK inhibition was associated with decreased hemolysis in response to diamide treatments or osmotic shock, and with decreased erythrocyte sickling under experimental hypoxia. These findings provided insights into MAPK-mediated signaling pathways that regulate erythrocyte function and hemolysis in response to extracellular stressors or human diseases.

Contribution of Stenotrophomonas maltophilia MfsC transporter to protection against diamide and the regulation of its expression by the diamide responsive repressor DitR.

Stenotrophomonas maltophilia contains an operon comprising mfsB and mfsC, which encode membrane transporters in the major facilitator superfamily (MFS). The results of the topological analysis predicted that both MfsB and MfsC possess 12 transmembrane helices with the N- and C-termini located inside the cells. The deletion of mfsC increased the susceptibility to diamide, a chemical oxidizing agent, but not to antibiotics and oxidative stress-generating substances relative to wild-type K279a. Moreover, no altered phenotype was observed against all tested substances for the ΔmfsB mutant. The results of the expression analysis revealed that the mfsBC expression was significantly induced by exposure to diamide. The diamide-induced gene expression was mediated by DitR, a TetR-type transcriptional regulator encoded by smlt0547. A constitutively high expression of mfsC in the ditR mutant indicated that DitR acts as a transcriptional repressor of mfsBC under physiological conditions. Purified DitR was bound to three sites spanning from position + 21 to -57, corresponding to the putative mfsBC promoter sequence, thereby interfering with the binding of RNA polymerase. The results of electrophoretic mobility shift assays illustrated that the treatment of purified DitR with diamide caused the release of DitR from the mfsBC promoter region, and the diamide sensing mechanism of DitR required two conserved cysteine residues, Cys92 and Cys127. This suggests that exposure to diamide can oxidize DitR through the oxidation of cysteine residues, leading to its release from the promoter, thus allowing mfsBC transcription. Overall, MfsC and DitR play a role in adaptive resistance against the diamide of S. maltophilia.

Toxicological and transcriptomic effects in Mythimna separata (Lepidoptera: Noctuidae) exposed to chlorantraniliprole and functional characterization of glutathione S-transferases.

BACKGROUND: Chlorantraniliprole (CAP) is an efficient anthranilic diamide insecticide against economically important pests such as the oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae). Resistance to CAP may develop due to enhanced enzymatic detoxification. The glutathione S-transferase (GST) superfamily in M. separata has not been systematically characterized. The aim of this study was therefore to explore the effects of lethal and sublethal doses of CAP on M. separata larvae, screen differentially expressed genes (DEGs) responding to CAP exposure, identify and characterize the GST superfamily, and analyze the metabolism of CAP by recombinant GSTs. RESULTS: The toxicity bioassay showed that CAP was active against M. separata third-instar larvae. LC50 was 17.615, 3.127, and 1.336 mg/L after 24, 48, and 72 h, respectively. Poisoned larvae showed contracted somites and disrupted midgut. Total GST activity in larvae was significantly elevated 24 h after CAP exposure. RNA-sequencing generated 43 055 unigenes with an average length of 1010 bp, and 567 up-regulated and 692 down-regulated DEGs responding to CAP treatment were screened. Thirty-five GST genes were identified from unigenes, including 31 cytosolic, three microsomal, and one unclassified. The expression profile of GST genes was analyzed using samples from different developmental stages, adult tissues, and CAP treatments. Metabolic assays indicated that CAP was depleted by recombinant MseGSTe2 and MseGSTs6. CONCLUSIONS: This study provides insight into the toxicological and transcriptomic effects in M. separata larvae exposed to CAP. The identification and functional characterization of the GST superfamily will improve our understanding of CAP detoxification by GSTs. © 2022 Society of Chemical Industry.

DDR1 promotes LoVo cell proliferation by regulating energy metabolism.

Cellular energy metabolism dysregulation is associated with colorectal cancer (CRC) development and progression. Discoidin domain receptor 1a (DDR1a), one of the five DDR1 isoforms, is closely related to cell proliferation, invasion, and apoptosis in various tumors. Whether it participates in cellular metabolic reprogramming and regulates CRC initiation and progression remains unclear. In this study, we compared the expression of DDR1 in CRC tissues and adjacent tissues from 126 postoperative CRC samples. Moreover, lentivirus-mediated DDR1a overexpression and knockdown were performed in LoVo cells, and cell viability and proliferation were determined by CCK-8 and BrdU assays, respectively. Oxygen consumption rate, extracellular acidification rate, and lactate production were used to determine the effect of DDR1a on metabolic reprogramming. Clinically, CRC patients with high DDR1 expression had poor differentiation and were at an advanced TNM stage. DDR1a promoted LoVo cell proliferation, mitochondrial function, and extracellular acidification. Moreover, DDR1a knockdown inhibited intracellular lactic acid production in LoVo cells, while a pyruvate kinase inhibitor (diamide, 200 µM) significantly reversed this progression. Taken together, our results reveal that DDR1 plays a crucial role in maintaining intracellular environment homeostasis through metabolic reprogramming.

Residue and distribution of drip irrigation and spray application of two diamide pesticides in corn and dietary risk assessment for different consumer groups.

BACKGROUND: As the use of diamide insecticides on corn continues to increase, there is growing concern about their residue levels on corn and dietary risks to populations. In this study, the distribution, dispersion and transfer efficiency of two diamide insecticides (tetrachlorantraniliprole (TCAP) and cyantraniliprole (CNAP)) in different parts of corn and soil were investigated in a 1-year field trial in Guangzhou and Lanzhou using two different application methods - spray and drip irrigation, respectively - and the dietary risk of the insecticides to different consumer populations was assessed under the two application methods. RESULTS: The results showed that drip irrigation had a longer persistence period than spraying, and there was a hysteresis in the absorption distribution of the agent in different parts of corn, which was gradually transferred to the leaves after absorption from the roots. The average TE1 (transfer efficiency) and TE2 were 0.230-0.261 and 1.749-1.851 for TCAP and 0.168-0.187 and 2.363-2.815 for CNAP, respectively. At corn harvest, both TCAP and CNAP were below detectable levels in soil and corn. For different consumer populations, hazard quotients ranged from 0.001 to 0.066 for TCAP and from 0.003 to 0.568 for CNAP - both well below 100%. CONCLUSION: This study indicates that TCAP and CNAP applied by spray or drip irrigation are safe for long-term risk of human intake and also provides guidance for the use of both insecticides in agricultural production to control corn pests, especially in arid and semi-arid areas. © 2022 Society of Chemical Industry.

Piperazine squaric acid diamides, a novel class of allosteric P2X7 receptor antagonists.

The P2X7 receptor (P2X7R) stands out among the purinergic receptors due to its strong involvement in the regulation of tumor growth and metastasis formation as well as in innate immune responses and afferent signal transmission. Numerous studies have pointed out the beneficial effects of P2X7R antagonism for the treatment of a variety of cancer types, inflammatory diseases, and chronic pain. Herein we describe the development of novel P2X7R antagonists, incorporating piperazine squaric diamides as a central element. Besides improving the antagonists' potency from pIC50 values of 5.7-7.6, ADME properties (logD7.4 value, plasma protein binding, in vitro metabolic stability) of the generated compounds were investigated and optimized to provide novel P2X7R antagonists with drug-like properties. Furthermore, docking studies revealed the antagonists binding to the allosteric binding pocket in two distinct binding poses, depending on the substitution of the central piperazine moiety.

Dichloro Butenediamides as Irreversible Site-Selective Protein Conjugation Reagent.

We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation.

Covalent inhibition of P. falciparum ferredoxin-NADP+ reductase: Exploring alternative strategies for the development of new antimalarial drugs.

The protozoan Plasmodium falciparum is the main aetiological agent of tropical malaria. Characteristic of the phylum is the presence of a plastid-like organelle which hosts several homologs of plant proteins, including a ferredoxin (PfFd) and its NADPH-dependent reductase (PfFNR). The PfFNR/PfFd redox system is essential for the parasite, while mammals share no homologous proteins, making the enzyme an attractive target for novel and much needed antimalarial drugs. Based on previous findings, three chemically reactive residues important for PfFNR activity were identified: namely, the active-site Cys99, responsible for hydride transfer; Cys284, whose oxidation leads to an inactive dimeric form of the protein; and His286, which is involved in NADPH binding. These amino acid residues were probed by several residue-specific reagents and the two cysteines were shown to be promising targets for covalent inhibition. The quantitative and qualitative description of the reactivity of few compounds, including a repurposed drug, set the bases for the development of more potent and specific antimalarial leads.

Involvement of plasma membrane H+-ATPase in diamide-induced extracellular alkalization by roots from pea seedlings.

MAIN CONCLUSION: The plasma membrane H+-ATPase can be considered as a redox-dependent enzyme, because diamide-mediated inhibition of its hydrolytic and transport activities is accompanied by alkalization of the rhizosphere and retardation of root growth. Plasma membranes were isolated from roots of etiolated pea seedlings treated in the presence of an oxidant-diamide and an inhibitor of redox-sensitive protein phosphatase-phenylarsine oxide. Hydrolytic and proton transport activities of H+-ATPase were determined. The effects of diamide appeared in inhibition of both ATP hydrolysis and the proton transport. However, root treatment with phenylarsine oxide only slightly reduced Vmax, but did not affect ATP-dependent proton transport. The thiol groups of cysteines in the proteins can act as molecular targets for both compounds. However, treatment of isolated membranes with diamide or dithiothreitol did not have any effect on the H+ transport. It can be assumed that water-soluble diamide acts indirectly and its effects are not associated with oxidation of H+-ATPase cysteines. Therefore, plasmalemma was subjected to PEGylation-process where reduced cysteines available for PEG maleimide (5 kDa) were alkylated. Detection of such cysteines was carried out by Western blot analysis with anti-ATPase antibodies. It was found that shifts in the apparent molecular weight were detected only for denaturated proteins. These data suggest that available thiols are not localized on the enzyme surfaces. BN-PAGE analysis showed that the molecular weights of the ATPase complexes are almost identical in all samples. Therefore, oligomerization is probably not the reason for the inhibition of ATPase activity. Roots treated with these inhibitors in vivo exhibited stunted growth; however, a strong alkaline zone around the roots was formed only in the presence of diamide. Involvement of H+-ATPase redox regulation in this process is discussed.

Whole-genome sequencing to detect mutations associated with resistance to insecticides and Bt proteins in Spodoptera frugiperda.

The fall armyworm (FAW), Spodoptera frugiperda, is a major pest native to the Americas that has recently invaded the Old World. Point mutations in the target-site proteins acetylcholinesterase-1 (ace-1), voltage-gated sodium channel (VGSC) and ryanodine receptor (RyR) have been identified in S. frugiperda as major resistance mechanisms to organophosphate, pyrethroid and diamide insecticides respectively. Mutations in the adenosine triphosphate-binding cassette transporter C2 gene (ABCC2) have also been identified to confer resistance to Cry1F protein. In this study, we applied a whole-genome sequencing (WGS) approach to identify point mutations in the target-site genes in 150 FAW individuals collected from China, Malawi, Uganda and Brazil. This approach revealed three amino acid substitutions (A201S, G227A and F290V) of S. frugiperda ace-1, which are known to be associated with organophosphate resistance. The Brazilian population had all three ace-1 point mutations and the 227A allele (mean frequency = 0.54) was the most common. Populations from China, Malawi and Uganda harbored two of the three ace-1 point mutations (A201S and F290V) with the 290V allele (0.47-0.58) as the dominant allele. Point mutations in VGSC (T929I, L932F and L1014F) and RyR (I4790M and G4946E) were not detected in any of the 150 individuals. A novel 12-bp insertion mutation in exon 15 of the ABCC2 gene was identified in some of the Brazilian individuals but absent in the invasive populations. Our results not only demonstrate robustness of the WGS-based genomic approach for detection of resistance mutations, but also provide insights for improvement of resistance management tactics in S. frugiperda.