Enzyme Activity Assays Research Articles

Effect of O. ficus-Indica endorhizosphere-associated- bacteria on the survival and mortality of carmine cochineal (Dactylopius opuntiae)

ABSTRACT The false carmine cochineal scale, Dactylopius opuntiae (Cockerell), Hemiptera: Dactylopiidae, is the most aggressive pest of Opuntia ficus-indica (L.) Mill, in many countries. The aim of this study is to evaluate the efficacy of culturable endorhizosphere bacteria isolated from the root of this cactus in terms of their potential antagonism as a biological control agent against this pest under laboratory and greenhouse conditions. Among 62 bacteria isolated from the cactus O. ficus-indica endorhizosphere, four bacterial isolates exhibited the highest insecticidal activities against nymphs and adult females of D. opuntiae. Under laboratory conditions, bacterial solutions of the strain ‘BME8’ Bacillus halotolerans and ‘Bm3C’ Priestia aryabhattai were sprayed onto D. opuntiae at concentrations 107 and 108 CFU.mL−1, respectively, which caused 100% mortality in adult females and nymphs within 24 h of treatment. While in greenhouse conditions, spraying the bacterial solution of the BME8 strain at 109 CFU.mL−1 on the covered cochineals, seemed to be the most effective, the number of insects declined by 59% after 7 days of application. The strain ‘Bm4D’ Bacillus licheniformis and ‘Bm1A’ Bacillus subtilis, played a key role in hydrolyzing the hydrophobic wax protecting females, which is the most important tool in controlling against D. opuntiae females. Furthermore, the strain ‘Bm1A’ Bacillus subtilis spray, was considered the second most efficient treatment in the greenhouse. The survival rate was 55% at 109 CFU.mL−1 after only 7 days of treatment. Enzymatic activity assays showed that all strains BME8, Bm1A and Bm4D (except Bm3C) exhibited α-amylase, pectinase and chitinase activities.

Two detoxification enzyme genes, CYP6DA2 and CarFE4, mediate the susceptibility to afidopyropen in Semiaphis heraclei

IntroductionSemiaphis heraclei is an important economic pest affecting Caprifoliaceae and Apiaceae plants, and chemical control is still the main effective control method in the field. Afidopyropen is a new type of pyridine cyclopropyl insecticide, which can effectively control piercing-sucking mouthparts pests and is suitable for pest resistance management. However, the detoxification mechanism of S. heraclei to afidopyropen is still poorly cleared.MethodsThe insecticidal activity of afidopyropen against S. heraclei and the enzyme activity assay and synergism bioassay were evaluated. The detoxification enzyme genes were obtained by transcriptome and validated by quantitative real-time PCR (RT-qPCR). Furthermore, RNA interference was used to study the functions of detoxification enzyme genes.ResultsThe activities of cytochrome P450 monooxygenases (P450s) and carboxylesterases (CarEs) were significantly increased under afidopyropen treatment. The toxicity of afidopyropen against S. heraclei was significantly increased after application the inhibitors of piperonyl butoxide and triphenyl phosphate. Sixteen P450 genes and three CarE genes were identified in the transcriptome of S. heraclei. The RT-qPCR results showed that eleven P450 genes and two CarE genes were significantly upregulated under afidopyropen treatment, and the expression of CYP6DA2 and CarFE4 was upregulated by more than 2.5 times. The expression pattern of CYP6DA2 and CarFE4 was further analyzed in different developmental stages of S. heraclei and knockdown of CYP6DA2 and CarFE4 significantly increased the susceptibility of S. heraclei to afidopyropen.ConclusionThe results of this study uncover the key functions of CYP6DA2 and CarFE4 in the detoxification mechanism of S. heraclei to afidopyropen, and provide a theoretical basis for the scientific use of afidopyropen in the field.

Differential Expression of Neurodegeneration-Related Genes in SH-SY5Y Neuroblastoma Cells Under the Influence of Cyclophilin A: Could the Enzyme be a Likely Trigger and Therapeutic Target for Alzheimer's Disease?

The function and mechanism of Cyclophilin A (CypA) in modulating gene expression associated with Alzheimer's disease (AD) remain unclear. This multifunctional protein is found to be elevated in the cerebrospinal fluid (CSF) of individuals at risk for AD. The cytotoxic effects of CypA, including both wild-type and the mutant R55A, were assessed using the MTT assay. Prior to this evaluation, the purified recombinant protein was validated through enzymatic activity assays and western blot analysis. Following treatment with CypA and transient transfection using the CypA construct, real-time PCR (qRT-PCR) and western blotting were conducted to analyze the expression of factors involved in various signaling pathways, with an emphasis on inflammation, cell death, and intercellular communication. The findings indicate that CypA has a significant impact on the gene expression of factors associated with inflammation and the progression of AD in SH-SY5Y cells. It can be concluded that CypA is capable of regulating gene expression in SH-SY5Y cells, either in a manner dependent on or independent of its enzymatic activity. Additionally, the influence of this multifunctional protein on gene expression is contingent upon the specific site of action, as well as the dosage and duration of exposure to the cells.

Proteomics analysis in rats reveals convergent mechanisms between major depressive disorder and dietary zinc deficiency

Abstract Background Preclinical and clinical studies have shown that dietary zinc deficiency can lead to symptoms similar to those observed in major depressive disorder (MDD). However, the underlying molecular mechanisms remain unclear. To investigate these mechanisms, we examined proteomic changes in the prefrontal cortex (PFC) and hippocampus (HP) of rats, two critical brain regions implicated in the pathophysiology of depression. Methods Rats were fed diets either adequate in zinc (ZnA, 50 mg Zn/kg) or deficient in zinc (ZnD, <3 mg/kg) for four weeks. High-throughput proteomic analysis was used to detect changes in protein expression, supplemented by enzyme activity assay for mitochondrial complexes I and IV, examining their functional impacts. Results ZnD led to significant alterations in protein expression related to zinc transport and mitochondrial function. Proteomic analysis revealed changes in zinc transporter family members such as Slc30a1 (6.64 log2FC), Slc30a3 (-2.32 log2FC), Slc30a4 (2.87 log2FC), Slc30a5 (5.90 log2FC), Slc30a6 (1.50 log2FC), and Slc30a7 (2.17 log2FC) in the PFC, and Slc30a3 (-1.02 log2FC), Slc30a5 (-1.04 log2FC), and Slc30a7 (1.08 log2FC) in the HP of rats subjected to ZnD. Furthermore, ZnD significantly affected essential mitochondrial activity proteins, including Atp5pb (3.25 log2FC), Cox2 (2.28 log2FC), Atp5me (2.04 log2FC), Cyc1 (2.30 log2FC), Cox4i1 (1.23 log2FC), Cox7c (1.63 log2FC), and Cisd1 (1.55 log2FC), with a pronounced decrease in complex I activity in the PFC. Conclusions Our study demonstrates that ZnD leads to significant proteomic changes in the PFC and HP of rats. Specifically, ZnD alters the expression of zinc transporter proteins and proteins critical for mitochondrial function. The significant decrease in complex I activity in the PFC further underscores the impact of ZnD on mitochondrial function. These results highlight the molecular mechanisms by which ZnD can influence brain function and contribute to symptoms similar to those observed in depression.

Genome-wide investigation of family‑1 UDP glycosyltransferases reveals the Fusarium resistance role of VfUGT90A2 in tung trees

UDP-glycosyltransferases (UGTs) play vital roles in pathogen resistance through secondary metabolites, such as flavonoid. Vernicia fordii (tung trees) is well known for its production of industrial oil, which has been decimated by the soil-borne fungus Fusarium oxysporum f. sp. Fordiis (Fof-1). The current understanding of the metabolic and molecular mechanism of V. fordii against Fof-1 is limited. Through genome-wide analysis identified 153 VfUGTs, which were divided into 16 clusters derived from tandem and segmental repeat events. Variations in gene structure and cis-elements have contributed to gene differentiation within the various subfamilies of VfUGTs. VfUGT90A2 was found to be highly induced and expressed, serving as a key gene during the response process to Fof-1 infection in tree roots. The study showed that transgenic hairy roots of the tung trees, overexpressing VfUGT90A2, displayed significantly enhanced resistance to Fof-1. Additionally, the root extract from these transgenic roots was found to have a notable inhibitory effect on the growth of Fof-1 mycelium. Metabolomic and transcriptomic analyses further revealed an increase in flavonoids, such as quercitrin and myricitrin, in the transgenic hairy roots overexpressing VfUGT90A2. Meanwhile, VfUGT90A2 was speculated bound well to quercetin by protein docking predictions. The enzyme activity assay in vitro further verified that VfUGT90A2 catalyzed the glycosylation of quercetin. In all, our data shows that VfUGT90A2 is involved in the flavonoid biosynthetic pathways and has an impact on enhancing the resistance of plant to Fusarium, therefore, offers an opportunity to explore an effective strategy for Fusarium resistant plant breeding.

Genome-wide identification and expression analysis of the GT8 gene family in tomato(Solanum lycopersicum) and the functional of SlGolS1 under cold stress

Glycosyltransferases (GTs) are a diverse superfamily of enzymes involved in glycosylation reactions, with the GT8 (glycosyltransferase 8) playing a crucial role in plant growth, development, and abiotic stress responses. Tomato, widely cultivated and is a thermophilic plant. So it is significant to study how GT8 regulates cold resistance in tomato for plant growth. In this study, we screened the whole genome of tomato by using bioinformatics methods and identified 40 members of the GT8 gene family. Analysis of cold stress transcriptome data and qRT-PCR experiments revealed the potential significance of SlGolS1 in responding to cold stress. SlGolS1 was highly expressed in the stems and flowers of tomato, with its mature protein localized in the chloroplast. Used the VIGS method to transiently silence the SlGolS1 gene, the SlGolS1-silenced plants (pTRV2-SlGolS1) rendered tomato more sensitive to cold stress compared with the control (pTRV2) tomato plant phenotype after cold treatment; enzyme activity assays showed that oxidative damage was more severe in the pTRV2-SlGolS1. In summary, SlGolS1 positively regulates cold resistance in tomato.

Disrupted NAD(P) Metabolism and Xanthine Dehydrogenase in a Stress-Induced Rat Model of Depression: NMR Metabolomics Insights

Background: Clinical findings have shown a negative correlation between the severity of depressive symptoms and serum uric acid levels in men, yet the role of metabolic regulation in the pathophysiology of depression remains largely unknown. Methods: In this study, we utilized an acute restraint-stress-induced male rat model of depression to investigate biochemical changes through NMR-based metabolomics combined with serum biochemical analysis. Additionally, we employed qPCR, immunoblotting, and enzyme activity assays to assess the expression and activity of xanthine oxidoreductase, the rate-limiting enzyme in uric acid production. Results: Our findings indicate the following: (1) restraint stress is a valid method for inducing a depressive phenotype in rats; (2) depressive rats exhibit decreased NAD(P) levels in the liver and increased nicotinamide N-oxide and nicotinate levels in urine, accompanied by decreased levels of uric acid, allantoin, and allantoic acid in serum or tissues; (3) xanthine dehydrogenase activity is diminished in depressive rats without corresponding changes in gene or protein expression. Conclusion: The increased urinary excretion of NAD(P) precursors results in reduced hepatic NAD(P) levels, thereby suppressing NAD-dependent xanthine dehydrogenase activity and diminishing the production of uric acid and its downstream metabolites (allantoin and allantoic acid).

Expression and functional study of DNA polymerases from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963.

The properties of DNA polymerases isolated from thermophilic and mesophilic microorganisms, such as the thermophilic Geobacillus stearothermophilus (Bst) and mesophilic Bacillus subtilis phage (Phi29), have been widely researched. However, DNA polymerases in psychrophilic microorganisms remain poorly understood. In this study, we present for the first time the expression and functional characterization of DNA polymerases PWT-WT and FWT-WT from Psychrobacillus sp. BL-248-WT-3 and FJAT-21963. Enzymatic activity assays revealed that FWT-WT possessed strand displacement but lacked exonuclease activity and high ionic strength tolerance, whereas PWT-WT lacked all these properties. Further protein engineering and biochemical analysis identified D423 and S490 as critical mutation sites for improving strand displacement and tolerance to high ionic strength, specifically in the presence of 0-0.3 M potassium chloride (KCl), sodium chloride (NaCl), and potassium acetate (KAc). Three-dimensional structural analysis demonstrated that the size and the electric charge of the single-stranded DNA (ssDNA) encapsulation entrance were pivotal factors in the binding of the ssDNA template.

In Vitro Biological Target Screening and Colloidal Aggregation of Minor Cannabinoids.

There is limited information on interactions between cannabinoids and many pharmacologically and toxicologically relevant targets in humans (e.g., receptors, ion channels, enzymes, and transporters). To address this data gap, seven cannabinoids were screened against a panel of 44 safety-related biological targets in competitive ligand binding or enzymatic activity assays. Diverse binding profiles were observed among the cannabinoids; however, colloidal aggregates were detected by dynamic light scattering and a detergent-sensitive enzyme inhibition assay. These aggregates may nonspecifically inhibit targets, yielding false positives. Although screening identified aggregates, additional testing is required to confirm cannabinoid aggregation in individual in vitro assays.

Proteomic Profiling of Venoms from Bungarus suzhenae and B. bungaroides: Enzymatic Activities and Toxicity Assessment.

Kraits are venomous snakes of the genus Bungarus from the family Elapidae. Their venom typically demonstrates neurotoxicity; however, the toxicity is significantly influenced by the snake's species and geographical origin. Among the Bungarus species, Bungarus suzhenae and B. bungaroides have been poorly studied, with little to no information available regarding their venom composition. In this study, a proteomic approach was employed using LC-MS/MS to identify proteins from trypsin-digested peptides. The analysis revealed 102 venom-related proteins from 18 distinct functional protein families in the venom of B. suzhenae, with the primary components being three-finger toxins (3-FTx, 25.84%), phospholipase A2 (PLA2, 40.29%), L-amino acid oxidase (LAAO, 10.33%), Kunitz-type serine protease inhibitors (KUN, 9.48%), and snake venom metalloproteinases (SVMPs, 6.13%). In the venom of B. bungaroides, 99 proteins from 17 families were identified, with primary components being 3-FTx (33.87%), PLA2 (37.91%), LAAO (4.21%), and KUN (16.60%). Enzymatic activity assays confirmed the presence of key venom enzymes. Additionally, the LD50 values for B. suzhenae and B. bungaroides were 0.0133 μg/g and 0.752 μg/g, respectively, providing a reference for toxicity studies of these two species. This research elucidates the proteomic differences in the venoms of these two species, offering a foundation for developing antivenoms and clinical treatments for envenomation.

Ailanthone targets the KMT2A-MEN1 complex to suppress lung metastasis of osteosarcoma

BackgroundLung metastasis is the leading cause of death in patients with osteosarcoma (OS), and new drugs are urgently needed. Epigenetic reprogramming is a recently proposed hallmark of malignancy; therefore, targeting epigenetic enzymes might provide a novel therapeutic strategy for OS lung metastasis. We recently reported that ailanthone (AIL), a natural product isolated from the Chinese medicinal plant Ailanthus altissima, inhibits OS cell growth and induces substantial metabolic changes; however, its direct targets remain unclear. PurposeTo identify the direct targets of AIL in OS and to explore the effects of AIL on OS lung metastasis in vivo. Study designDirect target proteins of AIL and downstream signaling pathways were identified in Saos-2 and U-2OS OS cells. The in vivo effects of AIL on OS lung metastasis were investigated using a mouse model. MethodsA novel surface plasmon resonance-high-performance liquid chromatography-mass spectrometry (SPR-HPLC-MS) assay was used to determine direct targets of AIL in OS. A cellular thermal shift assay, molecular docking analysis, enzyme activity assay, qRT-PCR, western blotting, chromatin immunoprecipitation assay, and reverse tests were performed to confirm the target and downstream pathway of AIL. A tumor xenograft model was used to verify the efficacy and mechanisms in vivo. ResultsHistone-lysine N-methyltransferase 2A (KMT2A) together with its scaffold protein menin (MEN1) were identified as direct target proteins of AIL in OS. AIL induced the autophagic degradation of the KMT2A-MEN1 complex. Moreover, AIL inhibited intracellular H3K4 methyltransferase activity and epigenetically inhibited the transcription of genes in the serine biosynthetic pathway (SSP). Furthermore, AIL suppressed OS lung metastasis and downregulated KMT2A, MEN1, and SSP in mouse models. ConclusionThis work showed that AIL targets the KMT2A-MEN1 complex and inhibits SSP to suppress OS lung metastasis. Notably, AIL exhibits new mechanisms of action, distinct from those of existing anti-OS drugs. On the basis of these findings, we proposed a novel strategy to treat OS by targeting epigenetic enzymes and cancer metabolism.

Honey-fried licorice in the treatment of arrhythmia: Structure elucidation and the mechanism of antiarrhythmic activity

Aim of the studyTo evaluate the therapeutic mechanism of Honey-fried licorice on arrhythmia, to explore the distribution of main components of Honey-fried licorice in vivo before and after processing, and to elucidate the active ingredient of Honey-fried licorice on arrhythmia. Materials and methodsUPLC-Q-TOF/MS were used to analyze the common and different components of raw and honey-fried licorice before and after processing. Yin deficiency syndrome was established by continuous irritability and water platform sleep deprivation, and then ventricular arrhythmia model was established by injection of calcium chloride into the tail vein. Applying the electrocardiograph changes in heart rate in rats. Subsequently, ELISA and histopathological examinations were conducted to assess the therapeutic effects of honey-fried licorice on arrhythmia. Metabonomics analysis was employed to predict key regulatory pathways involved in the treatment response. Finally, RT-PCR and enzyme activity assays were utilized to verify the expression and function of key genes and proteins, providing insights into the underlying mechanisms. ResultsThe heart rate of rats increased after injection of Cacl2 solution into the tail vein. Honey-fried licorice has a certain improvement effect on heart injury and tachycardia, and its mechanism may be through the obvious correction effect on SOD, MDA, LDH, Na+-K+-ATPase, CaM and CAMK2 in the arrhythmia model. Under pathological conditions, Metabonomics revealed that the heart was highly exposed to glycyrrhetic acid 3-O-glucuronide, isoformononetin, araboglycyrrhizin, 18β-glycyrrhetinic acid, liquiritigenin, licoflavonol and isoliquiritigenin are known to have anti-arrhythmic effects through immune regulation and oxidation. Notably, both PCR and ELISA analyses indicated that honey-fried licorice may effectively treat arrhythmia, potentially through the modulation of the arachidonic acid pathway. ConclusionThese results suggested that honey-fried licorice could protect against arrhythmia and alleviate oxidative stress and tissue damage caused by arrhythmia. Through correlation analysis and metabolomics, it was found that glycyrrhetic acid 3-O-glucuronide, isoformononetin, araboglycyrrhizin, 18β-glycyrrhetinic acid, liquiritigenin, licoflavonol and isoliquiritigenin can be used as the active ingredient of honey-fried licorice in the treatment of arrhythmia. Moreover, our results suggested that the therapeutic effect of honey-fried licorice on arrhythmia may be linked to the regulation of the arachidonic acid pathway. This study elucidates the mechanisms by which honey-fried licorice treats arrhythmia from a metabolic perspective, highlighting its role in "tonifying the spleen and stomach, supplementing qi, and replenishing the pulse." These findings provide a foundation for the further application of honey-fried licorice and the development of related products.

Up-regulated succinylation modifications induce a senescence phenotype in microglia by altering mitochondrial energy metabolism

The aging of the central nervous system(CNS) is a primary contributor to neurodegenerative diseases in older individuals and significantly impacts their quality of life. Neuroinflammation, characterized by activation of microglia(MG) and release of cytokines, is closely associated with the onset of these neurodegenerative diseases. The activated status of MG is modulated by specifically programmed metabolic changes under various conditions. Succinylation, a novel post-translational modification(PTM) mainly involved in regulating mitochondrial energy metabolism pathways, remains unknown in its role in MG activation and aging. In the present study, we found that succinylation levels were significantly increased both during aging and upon lipopolysaccharide-induced(LPS-induced) MG activation undergoing metabolic reprogramming. Up-regulated succinylation induced by sirtuin 5 knockdown(Sirt5 KD) in microglial cell line BV2 resulted in significant up-regulation of aging-related genes, accompanied by impaired mitochondrial adaptability and a shift towards glycolysis as a major metabolic pathway. Furthermore, after LPS treatment, Sirt5 KD BV2 cells exhibited increased generation of reactive oxygen species(ROS), accumulation of lipid droplets, and elevated levels of lipid peroxidation. By employing immunoprecipitation, introducing point mutation to critical succinylation sites, and conducting enzyme activity assays for succinate dehydrogenase(SDH) and trifunctional enzyme subunit alpha(ECHA), we demonstrated that succinylation plays a regulatory role in modulating the activities of these mitochondrial enzymes. Finally, down-regulation the succinylation levels achieved through administration of succinyl phosphonate(SP) led to amelioration of MG senescence in vitro and neuroinflammation in vivo. To our knowledge, our data provide preliminary evidence indicating that up-regulated succinylation modifications elicit a senescence phenotype in MG through alterations in energy metabolism. Moreover, these findings suggest that manipulation of succinylation levels may offer valuable insights into the treatment of aging-related neuroinflammation.Graphical abstracts

Profiling Secondary Metabolites of Governor’s Plum (Flacourtia indica Burm.f. Merr.) Fruit and their Potential Antioxidant and Chemopreventive Values

Objective: The goal of this study was to profile the secondary metabolites of two ripe -light and dark- stages of Flacourtia indica fruit and the assessment of their antioxidant and ant-proliferative activities. Methods: Secondary metabolites were profiled by GC-MS. Radical scavenging activity and scavenging of H2O2 assays were used to assess the antioxidant activities, while cell viability and CYP1B1 enzyme activity assays were used to assess the antiproliferative activities of the fruit extracts. Results: One hundred and twenty-three (123) metabolites were identified. DPPH activity showed a dose-dependent response to fruit extracts, and H2O2 scavenging activity increased with concentrations. Anti-proliferative properties using the MTS assay on prostate, breast and colon cancer cells and normal prostate cell lines revealed a relatively weak impact on the examined cells. DCM extracts exhibited inhibition of the activity of human cytochrome P450 1B1 enzyme. Conclusion: Results showed that fruit extracts demonstrated significant antioxidant activity, while they did not show cytotoxic effects on normal or cancer cells. DCM and hexane extracts showed noticeable inhibition of CYP1B1 enzyme, suggestive of a chemoprotective potential.

A comprehensive analysis of the defense responses of Odontotermes formosanus (Shiraki) provides insights into the changes during Serratia marcescens infection

BackgroundOdontotermes formosanus (Shiraki) is a highly damaging agroforestry pest. Serratia marcescens is a broad-spectrum insecticidal pathogen and is highly lethal to O. formosanus. However, little is known about the mechanism between them. To improve the biological control of pests, a more in-depth analysis of the interactions between the pests and the pathogens is essential.ResultsWe used RNA-seq, enzyme activity assays and real-time fluorescent quantitative PCR (qPCR) to explore the defense responses of O. formosanus against SM1. RNA-seq results showed that 1,160, 2,531 and 4,536 genes were differentially expressed at 3, 6 and 12 h after SM1 infection, and Kyoto Encyclopedia of Genes and Genomes (KEGG) results indicated that immune response and energy metabolism were involved in the defense of O. formosanus against SM1. Reactive oxygen species (ROS) levels and ROS synthesis genes were significantly elevated, and the antioxidant system were induced in O. formosanus after SM1 infection. In addition, the cellular immune genes were affected, and the Toll, immune deficiency (Imd), Janus kinase/signal transducer and activator of transcription (JAK/STAT), c-Jun N-terminal Kinase (JNK) and melanization pathways were activated. In vitro, Oftermicin, an antimicrobial peptide, had a significantly inhibitory effect on SM1. Furthermore, the expression levels and enzyme activities of phosphofructokinase (PFK), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) in glycolysis and tricarboxylic acid (TCA) cycles were increased.ConclusionsOur results clearly demonstrated that O. formosanus defended against SM1 by activating the antioxidant system, innate immunity and energy metabolism. This study would provide useful information for the development of biological controls of O. formosanus.

A Detailed Proteomics and Metabolomics Landscape Sheds Light on the Mechanistic Insights Into the Resistance Response of Transgenic Pigeon Pea Against Wilt Stress.

Pigeon pea, vital for farmers in semi-arid regions, suffers yield losses from Fusarium wilt caused by Fusarium udum. This study demonstrates that introducing the rice oxalate oxidase 4 (Osoxo4) gene significantly boosts wilt resistance. Enhanced resistance in transgenic lines was confirmed through gene expression analysis, enzyme activity assays, biochemical assessments, histochemical staining and in vitro and in vivo bioassays, including spore germination tests. We performed proteomics and metabolomics analyses to investigate mechanisms of enhanced resistance. LC-MS/MS-based label-free proteomics of wilt-infected transgenic and wild-type pigeon pea leaves identified 2386 proteins, with 1048 showing significant abundance changes-738 upregulated and 310 downregulated-in transgenic plants. Notably, proteins such as HMG1/2-like protein, Putative nucleosome assembly protein C364.06, DEAD-box ATP-dependent RNA helicase 3, Lipoxygenase 1, Annexin D1 and Annexin-like protein RJ4 were significantly upregulated, indicating their potential role in developing wilt-resistant cultivars. Metabolomic analysis showed elevated levels of amino acids, sugars, oxalic acid, sugar alcohols and myo-inositol in transgenic pigeon pea, with upregulated pathways in Sugar and Starch Metabolism and Inositol Phosphate Metabolism, indicating enhanced resilience to wilt stress. This study highlights unique regulatory proteins and metabolites, offering insights into stress adaptation and guiding genetic interventions for breeding disease-resistant pigeon pea varieties.

Metabolomic and enzymatic markers reveal critical air exposure threshold for Crassostrea hongkongensis quality

Post-harvest air exposure is unavoidable during oyster transportation and storage, yet the physiological tolerance limits and underlying metabolic responses of commercially important oyster species remain poorly understood. While previous studies have focused on immediate post-harvest quality changes, there is limited knowledge about the time-dependent metabolic adaptations that determine product quality during extended air exposure. This study investigated the physiological and metabolic responses of Crassostrea hongkongensis during air exposure at 4 °C, focusing on identifying the optimal period for quality preservation. Using a combination of survival analysis, enzyme activity assays, and metabolomic profiling, we examined oysters exposed to air for up to 18 days, with particular emphasis on the critical first three days. Survival analysis showed 100 % survival rate at 4 °C through day 7, with mortality beginning thereafter, compared to significant mortality observed at 25 °C (complete mortality by day 7) and 37 °C (complete mortality by day 2). Analysis of antioxidant enzyme activities revealed complex, time-dependent changes, with robust responses observed within the first three days, indicating effective stress management. Metabolomic analysis identified 38 differentially abundant metabolites throughout the exposure period. Notably, the metabolic profile at day 3 showed a tendency to revert towards the control state, suggesting a temporary adaptive response. Key findings included stability in total antioxidant capacity (T-AOC) levels during the initial three days and subtle changes in flavor-related compounds, such as slight decreases in glutamate and aspartate levels. Correlation analyses revealed intricate interactions between enzyme activities and metabolites, highlighting complex stress response mechanisms. The relationship between T-AOC and key osmolytes underscored their critical role in maintaining cellular redox balance during the initial exposure period. Our findings suggest that the optimal window for maintaining C. hongkongensis quality during air exposure at 4 °C is within the first three days. During this period, oysters demonstrate effective adaptive responses, maintaining key quality attributes and nutritional value. Beyond this timeframe, the risk of quality degradation increases significantly. These results have important implications for the oyster industry, providing evidence-based guidelines for post-harvest handling, transportation, and storage practices. We recommend limiting air exposure during cold storage to no more than 3 days to ensure optimal product quality and consumer satisfaction.

Co-expression of α-L-rhamnosidase and β-glucosidase in Aspergillus Niger and its application in enzymatic production of quercetin

Quercetin (3,3′,4′,5,7-pentahydroxy flavonoid) is a bioactive flavonoid with significant medicinal properties. Similarly, Rutin (quercetin-3-O-rutinoside or 3′,4′,5,7-tetrahydroxy-flavone-3-rutinoside) is widely recognized flavonoid, and is converted to quercetin by α-L-rhamnosidase and β-D-glucosidase. In this study, engineered Aspergillus niger co-expressing α-L-rhamnosidase and β-glucosidase was successfully constructed, and engineered strain Rha1-BGL8 was obtained by enzyme activity assay. When submerged fermentation after 120 h in 5 L of fermenter, the α-L-rhamnosidase activity was 36.90 U/ml and β-glucosidase activity was 61.02 U/mL. The fermented complex enzyme solution was employed for the hydrolysis of rutin to quercetin, and the yield of quercetin reached 95.31 % after 48 h, molecular structure of quercetin was verified by highly specific chromatographic methods. This study lays a technical foundation for the potential industrial application of enzyme-catalyzed conversion of rutin to quercetin.

Hepatic enzyme induction and its potential effect on thyroid hormone metabolism in the metamorphosing tadpole of Xenopus laevis (African clawed frog).

Hepatic enzyme induction, an inherent defense system against xenobiotics, is known to simultaneously affect endocrine system functions in mammals under specific conditions, particularly thyroid hormone (TH) regulation. While this phenomenon has been studied extensively, the pathway leading to this indirect thyroid effect in mammals has unclear applicability to amphibians, despite the importance of amphibian species in assessing thyroid-disruptive chemicals. Here, we investigated the effects of three well-known mammalian enzyme inducers-β-naphthoflavone (BNF), pregnenolone carbonitrile (PCN), and sodium phenobarbital (NaPB)-on the gene expression of phase-I and phase-II metabolizing enzymes in Xenopus laevis tadpoles. Waterborne exposure to BNF and PCN significantly induced the expression of both phase-I (cytochrome P450, CYP) and phase-II enzymes (UDP-glucuronosyltransferase, UGT and sulfotransferase, SULT), but in different patterns, while NaPB exposure induced CYP2B expression without affecting phase-II enzymes in tadpoles, in contrast to mammals. Furthermore, an ex vivo hepatic enzyme activity assay confirmed that BNF treatment significantly increased phase-II metabolic activity (glucuronidation and sulfation) toward TH. These results suggest the potential for certain mammalian enzyme inducers to influence TH clearance in X. laevis tadpoles. Our findings provide insights into the profiles of xenosensing activity and enzyme induction in amphibians, which can facilitate a better understanding of the mechanisms of indirect effects on the thyroid system via hepatic enzyme induction in nonmammalian species.

Evaluating the estrogen degradation potential of laccase and peroxidase from Bacillus ligniniphilus L1 through integrated computational and experimental approaches

This study investigated the degradation potential of laccase and catalase-peroxidase from the extremophilic marine bacterium Bacillus ligniniphilus L1 against endogenous and synthetic estrogen compounds using an integrated computational and experimental approach. Molecular docking identified five estrogen compounds exhibiting reliable bindings with enzymes, which were then subjected to enzyme activity assays. The degradation potential of the two enzymes against five selected estrogen compounds were investigated and compared with their commercial counterparts. Laccase from L1 showed higher degradation potential against estrone (47.02 % without and 62.21 % with ABTS) compared to commercial laccase (39 % without and 54.20 % with ABTS). For estradiol valerate, commercial laccase showed a slightly higher degradation (52.47 %) than L1 laccase (49.94 %), but with ABTS, L1 laccase performed better (74.15 % vs. 68.03 %). Notably, L1 catalase-peroxidase demonstrated significantly higher degradation for all tested compounds compared to its commercial counterpart with efficiencies of 96.16 %, 89.09 %, 74.94 %, 64.91 %, and 62.80 % against estropipate, quinestrol, estradiol valerate, estriol and estrone, respectively, revealing its potential for commercial applications. Molecular dynamics simulations revealed the interaction and stability of enzyme-estrogen complexes, with MMGBSA binding energy calculations supporting experimental results. These findings highlight the usefulness of the computational approach in elucidating the molecular mechanisms underlying enzyme-mediated bioremediation of environmental contaminants.