Carboxylesterase Enzymes Research Articles

The ability of selected fungal strains to produce carboxylesterase enzymes for biodegradation and use of bifenthrin insecticide as carbon source: in vitro and in silico approaches.

Bifenthrin (BF) is a broad-spectrum type I pyrethroid insecticide that acts on insects by impairing the nervous system and inhibiting ATPase activity, and it hastoxic effects on non-target organisms and high persistence in the environment. This study aimed to determine the potential of six different fungi, including Pseudozyma hubeiensis PA, Trichoderma reesei PF, Trichoderma koningiopsis PD, Purpureocillium lilacinum ACE3, Talaromyces pinophilus ACE4, and Aspergillus niger AJ-F3, to degrade BF. Three different concentrations of BF, including 0.1%, 0.2%, and 0.3% w/v, were used in the sensitivity testing that revealed a significant (p ≤ 0.01) impact of BF on fungal growth. Enzymatic assays demonstrated that both intracellular and extracellular carboxylesterases hydrolyzed BF with the enzymatic activity of up to 175 ± 3 U (μmol/min) and 45 ± 1 U, respectively. All tested fungi were capable of utilizing BF as a sole carbon source producing 0.06 ± 0.01 to 0.45 ± 0.01mg dry biomass per mg BF. Moreover, the presence of PytH was determined in the fungi using bioinformatics tools and was found in A. niger, T. pinophilus, T. reesei, and P. lilacinum. 3D structures of the PytH homologs were predicted using AlphaFold2, and their intermolecular interactions with pyrethroids were determined using MOE. All the homologs interacted with different pyrethroids with a binding energy of lesser than -10kcal/mol. Based on the study, it was concluded that the investigated fungi have a greater potential for the biodegradation of BF.

Identification and biophysical characterization of potential phytochemical inhibitors of carboxyl/choline esterase from Helicoverpa armigera for advancing integrated pest management strategies

In the realm of disease vectors and agricultural pest management, insecticides play a crucial role in preserving global health and ensuring food security. The pervasive use, particularly of organophosphates (OPs), has given rise to a substantial challenge in the form of insecticide resistance. Carboxylesterases emerge as key contributors to OP resistance, owing to their ability to sequester or hydrolyze these chemicals. Consequently, carboxylesterase enzymes become attractive targets for the development of novel insecticides. Inhibiting these enzymes holds the potential to restore the efficacy of OPs against which resistance has developed. This study aimed to screen the FooDB library to identify potent inhibitory compounds targeting carboxylesterase, Ha006a from the agricultural pest Helicoverpa armigera. The ultimate objective is to develop effective interventions for pest control. The compounds with the highest scores underwent evaluation through docking studies and pharmacophore analysis. Among them, four phytochemicals—donepezil, protopine, 3′,4′,5,7-tetramethoxyflavone, and piperine—demonstrated favorable binding affinity. The Ha006a-ligand complexes were subsequently validated through molecular dynamics simulations. Biochemical analysis, encompassing determination of IC50 values, complemented by analysis of thermostability through Differential Scanning Calorimetry and interaction kinetics through Isothermal Titration Calorimetry was conducted. This study comprehensively characterizes Ha006a-ligand complexes through bioinformatics, biochemical, and biophysical methods. This investigation highlights 3′,4′,5,7-tetramethoxyflavone as the most effective inhibitor, suggesting its potential for synergistic testing with OPs. Consequently, these inhibitors offer a promising solution to OP resistance and address environmental concerns associated with excessive insecticide usage, enabling a significant reduction in their overuse.

Design, Synthesis, and Antifeedant Activity Evaluation of 13/14-Arylthioether Matrine Derivatives.

Introducing a sulfur atom into active agricultural molecules is an important strategy for pesticide development. Matrine, an environmentally friendly botanical pesticide, has the advantage of being easily degraded and has drawn attention in the agricultural field. To explore the novel matrine-type pesticides, in this study, we designed and synthesized 13/14-arylthioether matrine derivatives by introducing various aryl sulfide motifs into bioactive matrine. Most of the synthesized arylthioether matrines exhibited good antifeedant activity against Spodoptera exigua. Among them, compound 2q showed the best antifeedant effect with an EC50 value of 0.038 mg/mL, which is approximately 125-fold more activity than matrine and reached the activity level of commercial standard azadirachtin A. Furthermore, compound 2q exhibited an inhibitory effect on antifeedant-related enzyme carboxylesterase (CarE) from S. exigua. In short, the high activity of arylthioether matrines offers new insights into developing new antifeedants.

Sublethal effects of nitenpyram on the development of silkworm

The utilization of nitenpyram for aphid and whitefly control may induce environmental contamination and negative repercussions on non-target organisms. Formerly, we found that nitenpyram would pollute the peripheral and sub-peripheral areas of the adjacent mulberry orchard. Under acute toxicity conditions, nitenpyram induced oxidative damage in silkworms, affected biological metabolism, synthesis, immunity, and signal transduction. Considering the impact of nitenpyram mist drift on mulberry leaves, we investigated the effects of low concentrations of nitenpyram on silkworms. The results showed that silkworms exposed to 0.17 mg/L, 0.35 mg/L and 0.70 mg/L of nitenpyram (1/40 LC50, 1/20 LC50 and 1/10 LC50) showed obvious poisoning symptoms. The cocoon weight and cocoon shell weight decreased gradually with increases in the concentration, and these decreases prolonged the growth and development time of silkworms and induced the detoxification enzymes carboxylesterase (CarE) and glutathione-S-transferase (GST) to cope with the stress damage caused by nitenpyram. Exposure to low concentrations of nitenpyram downregulates genes involved in the drug metabolism-other enzymes and peroxisome pathway in silkworms. Additionally, through injection of miRNA mimics and inhibitors, we discovered that detoxifying enzyme pathway genes are influenced by bmo-miR-3382-3P, bmo-miR-3213-5P and bmo-miR-133, regulating the immune response of silkworms. This study provides an overall view of the toxicity and detoxification metabolism of nitenpyram in silkworm, and provides a reference for environmental assessment.

Enzyme-based sensor for the real-time detection of atrazine: Evidence from electrochemical and docking studies

This study focused on strategically employing the carboxylesterase enzyme Ha006a, derived from the pesticide-resistant microorganism Helicoverpa armigera, to detect atrazine. A comprehensive analysis through biochemical, biophysical and bioinformatics approaches was conducted to determine the interaction between the Ha006a protein and the herbicide atrazine. These experimental findings elucidated the potential of leveraging the inherent pesticide sequestration mechanism of the Ha006a enzyme for sensor fabrication. Numerous optimizations were undertaken to ensure the precision, reproducibility and convenient storage of the resulting electrochemical sensor, Ha006a/MCPE. This biosensor exhibited exceptional performance in detecting atrazine, demonstrating outstanding selectivity with a lower limit of detection of 5.4 µM. The developed biosensor has emerged as a reliable and cost-effective green tool for the detection of atrazine from diverse environmental samples. The Ha006a-based biosensor fabrication has expanded the possibilities for the efficient integration of insect enzymes as analytical tools, paving the way for the design of cost-effective biosensors capable of detecting and quantifying pesticides.

Determination of target site mutations and detoxification enzymes in the abamectin resistance of spider mite Tetranychus urticae collected from cotton fields of Türkiye

The two-spotted spider mite, Tetranychus urticae (Koch, 1836) (Acari: Tetranychidae) causes significant problems in cotton fields. The pest has developed resistance to abamectin, which is used intensely against spider mite populations. A discriminating dose was applied to T. urticae populations collected from Aydın, Adana, Şanlıurfa, and Diyarbakır provinces between 2019 and 2021, where there is significant cotton production and intensive acaricide applications. Populations with a mortality rate of less than 80% at the discriminating dose applied to field populations were included in the bioassay study. Then, the 10 most resistant populations were determined based on their LC50 and LC90 values. It was observed that there was high resistance (HR) in two T. urticae populations collected from Diyarbakır (DIY28 and DIY2) with LC50 values of 24.5 and 33.81 mg L-1, respectively 113 and 156-fold and one population collected from the province of Aydın (AYD4) LC50 value of 23.5 mg L-1 and 109-fold. As a result of the comparisons with a susceptible population in biochemical studies, it was determined that the carboxylesterase (CarE) enzyme of these populations (except for SAN6, SAN7, SAN8, ADA9, and ADA11) had more than 2-fold higher activity compared to the glutathione enzyme (GST), and they were in statistically different groups. When the GST enzyme activities were examined, it was found that the enzyme activities were generally low, except for the ADA11 and ADA16 populations (3.73 and 2.3-fold). In terms of the target site resistance, it was observed that there was no target site mutation (G314D and I321T-G326E) in the glutamate-gated chloride channels (Units GluCl 1 and 3) in these highest 10 resistant populations. It can be said that the resistance levels determined for abamectin from the obtained populations were determined to be low. In brief, out of the total 44 populations brought in and tested, only 30 populations were considered resistant with a mortality rate below 80% after the applied discriminating dose. It was determined that 6.8% of these 30 populations had HR, 31.8% had medium resistance (MR), and 29.5% had low resistance (LR). When examining by regions, it was found that high resistance populations were more prevalent in Diyarbakır and Aydın (15.4% and 10%, respectively) provinces, while the highest percentage of MR populations was found in Şanlıurfa, at 90%. Abamectin still acts as an effective acaricide. However, it is recommended to use acaricides with different modes of action to prevent possible emergence of resistance cases.

Assessing the impact of antiviral drugs commonly utilized during the COVID-19 pandemic on the embryonic development of Xenopus laevis

The antiviral drugs favipiravir and oseltamivir are widely used to treat viral infections, including coronavirus 2019 (COVID-19), and their levels are expected to increase in the aquatic environment. In this study, the potential toxic and teratogenic effects of these drugs were evaluated using the frog embryo teratogenesis assay Xenopus (FETAX). In addition, glutathione S-transferase (GST), glutathione reductase (GR), catalase, carboxylesterase (CaE), and acetylcholinesterase (AChE) enzyme activities and malondialdehyde levels were measured as biochemical markers in embryos and tadpoles for comparative assessment of the sublethal effects of the test compounds. Prior to embryo exposure, drug concentrations in the exposure medium were measured with high-performance liquid chromatography. The 96-h median lethal concentration (LC50) was 137.9 and 32.3 mg/L for favipiravir and oseltamivir, respectively. The teratogenic index for favipiravir was 4.67. Both favipiravir and oseltamivir inhibited GR, CaE, and AChE activities in embryos, while favipiravir increased the GST and CaE activities in tadpoles. In conclusion, favipiravir, for which teratogenicity data are available in mammalian test organisms and human teratogenicity is controversial, inhibited Xenopus laevis embryo development and was teratogenic. In addition, sublethal concentrations of both drugs altered the biochemical responses in embryos and tadpoles, with differences between the developmental stages.

Lipid-Gelucire based rectal delivery of ramipril prodrug exhibits significant lowering of intra-ocular pressure in normotensive rabbit: sustained structural relaxation release kinetics and IVIVC

Carboxylesterase enzymes convert a prodrug ramipril into the biologically active metabolite ramiprilat. It is prescribed for controlling ocular hypertension after oral administration. High concentrations of carboxylesterase enzymes in rectal and colon tissue can transform ramipril significantly to ramiprilat. Sustained rectal delivery of ramipril has been developed for intra-ocular pressure lowering effect using a normotensive rabbit model. Rectal suppositories have been formulated using a matrix base of HPMC K100-PEG 400-PEG 6000, incorporating varying amounts of Gelucire by the fusion moulding method. The presence of Gelucire in the suppository exhibited sustained structural relaxation-based release kinetics of RM compared to its absence. Intravenous and oral administration of ramipril has decreased IOP in the treated rabbit up to 90 and 360 min, respectively. Treated rabbits with suppositories have revealed decreased IOP for an extended period compared to the above. Formulation containing GEL 3% reduced intra-ocular pressure to 540 min, with the highest area under the decreased IOP curve. Compared to oral, the pharmacodynamic bioavailability of ramipril has been improved significantly using a sustained-release rectal suppository. A rectal suppository for sustained delivery of ramipril could be used to lower IOP significantly.

Metabolite Formation of Pesticide Isoprocarb in Coffee Beans During Short-Term Storage and Condition

Isoprocarb (IPC) is one of the most important carbamate pesticides for white flea control in coffee plants. The prevalence of isoprocarb pesticide residues in coffee, i.e., green coffee beans, is a cause for concern. Green coffee beans were intentionally contaminated with isoprocarb at concentrations >0.01 mg/kg, which was investigated in this research. Quantitative analysis of isoprocarb with the QuEChERS method and qualitative analysis for metabolite formation was characterized using UV-Vis, FTIR, 1H-NMR, GC-MS, and LC-QTOF-MS. Based on the data, the metabolite formed is o-cumenol through the hydrolysis reaction of o-aryl carbamate with the enzyme carboxylesterase. o-cumenol or 2- isopropylphenol as a phenol derivative. To verify the existence of metabolite analytes that are believed to be there in coffee tainted with isoprocarb, more reliable analytical techniques utilizing analytical standards must be developed. Isoprocarb concentrations decreased along the storage time, especially in unsterilized conditions. As a toxic compound, a quantitative structural activity relationship study (QSAR) was initially carried out through a software application for estimating toxicity (TEST) provided by the US Environmental Protection Agency (EPA). For additional information, based on the LC50 and LD50 data confirmed from the TEST application, it was concluded that isoprocarb is more toxic than o-cumenol.

Activity of carboxylesterase enzyme in different tissues of sixth instar larvae Orthaga exvinacea Hampson under various conditions of temperature and pH

The mango leaf-webber, Orthaga exvinacea Hampson is one of the major pests of mango crop. In larval stage, they defoliate the leaves and thereby reduce the crop yield but in adult stage, they do not cause any damage to the crop. Insect must chemically alter and dispose of a large variety of compounds to maintain their normal body function. They must neutralise, inactivate or eliminate many foreign substances including pesticides. All foreign organic compounds are susceptible to metabolic attack in vivo. This biotransformation may involve by the activity of some enzymes. Carboxylesterases are major enzymes involved in the resistance process in several insect species. The appraisal and hydrolytic capacity of these catalysts work best inside particular temperature and hydrogen ion concentration (pH) conditions. The impacts of temperature and pH on the hydrolysis of both α-naphthyl acetate and β-naphthyl acetate by Orthaga exvinacea carboxylesterase and the tissue fractionation of these enzymes were examined. The ideal conditions for measuring carboxylesterase movement of Orthaga exvinacea were pH of 6.6 - 7.2 and temperature 35°C - 45°C. The particular carboxylesterase action indicates most extreme behaviour at pH 6.8 and temperature at 40oC. Tissue confinement investigation of the enzyme in Orthaga exvinacea demonstrated the nearness of carboxylesterase action in every division.

Bioactivity and Sublethal Effects of Ageratina adenophora (Asteraceae) on Bactrocera dorsalis (Diptera: Tephritidae)

Abstract Local fruit orchards have incurred heavy losses due to invasive insects, including the oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), in Guizhou Province, China. In our efforts to manage this invasive pest, a laboratory study was conducted to evaluate the response of adult B. dorsalis to an extract of Ageratina adenophora (Spreng.) R.M. King & H. Rob (Gunneridae: Asteraceae). We evaluated the contact toxicity and oviposition-deterrent activity of the extract on adult B. dorsalis, as well as the sublethal effects on survival and reproduction. We found that the A. adenophora extract has significant insecticidal activity, with the 24-h median lethal concentration (LC50) for adults being 26.014 mg/ml. The extract of A. adenophora also showed oviposition inhibition. A residual deterrent effect (28.16%) was evident up to 5 d after treatment. Exposure to the LC25 concentration of the A. adenophora extract caused significant differences in preoviposition and fecundity, decreased adult longevity in the F0 generation, and decreased egg and pupa survival in the F1 generation. The levels of carboxylesterase enzyme activity in adults treated with the extract were significantly lower than those in the untreated controls at 12 h and 24 h. This latter phenomenon could perhaps be an adaptive response to the extract that might reduce its toxic effects in B. dorsalis.

Unveiling the Antimicrobial and Larvicidal Potential of Butyrolactones and Orsellinic Acid Derivatives from the Morus alba-derived Fungus Aspergillus terreus via Integrated In vitro and In silico Approaches.

The emergence of multi-drug-resistant microbial strains spurred the search for antimicrobial agents; as a result, two distinct approaches were combined: four in vitro studies and four corresponding molecular docking investigations. Antituberculosis, anti-methicillin-resistant Staphylococcus aureus (anti-MRSA), antifungal, and larvicidal activities of the crude extract, two fractions, and seven isolated compounds from Aspergillus terreus derived from Morus alba roots were explored. The isolated compounds (5 butyrolactones and 2 orsellinic acid derivatives) showed potent to moderate antitubercular activity with MIC values ranging from 1.95 to 62.5 μg/mL (compared to isoniazid, 0.24 μg/mL) and promising anti-MRSA potential with inhibition zone diameters ranging from 8 to 25 mm. Additionally, the in silico study proved that the isolated compounds bind to the two corresponding proteins' active sites with high to moderate -(C-Docker interaction energies) and stable interactions. The isolated compounds displayed antifungal activities against different fungal strains at diverse degrees of activity, among them compound (8"S,9")-dihydroxy-dihydrobutyrolactone I eliciting the best antifungal activity. Meanwhile, all isolated compounds, fractions, and the crude extract demonstrated extremely selective potent to moderate activity against Cryptococcus neoformans. The isolated five butyrolactone derivatives could develop potential mosquito larvicidal agents as a result of promising docking outcomes in the larval enzyme carboxylesterase.

Insecticidal activity and underlying molecular mechanisms of a phytochemical plumbagin against Spodoptera frugiperda.

Plumbagin is an important phytochemical and has been reported to exhibit potent larvicidal activity against several insect pests, However, the insecticidal mechanism of plumbagin against pests is still poorly understood. This study aimed to investigate the insecticidal activities of plumbagin and the underlying molecular mechanisms against a devastating agricultural pest, the fall armyworm Spodoptera frugiperda. The effects of plumbagin on S. frugiperda larval development and the activities of two detoxification enzymes were initially examined. Next, transcriptomic changes in S. frugiperda after plumbagin treatment were investigated. Furthermore, RNA-seq results were validated by qPCR. Plumbagin exhibited a high larvicidal activity against the second and third instar larvae of S. frugiperda with 72h LC50 of 0.573 and 2.676mg/g, respectively. The activities of the two detoxification enzymes carboxylesterase and P450 were significantly increased after 1.5mg/g plumbagin treatment. Furthermore, RNA-seq analysis provided a comprehensive overview of complex transcriptomic changes in S. frugiperda larvae in response to 1.5mg/g plumbagin exposure, and revealed that plumbagin treatment led to aberrant expression of a large number of genes related to nutrient and energy metabolism, humoral immune response, insect cuticle protein, chitin-binding proteins, chitin synthesis and degradation, insect hormone, and xenobiotic detoxification. The qPCR results further validated the reproducibility and reliability of the transcriptomic data. Our findings provide a valuable insight into understanding the insecticidal mechanism of the phytochemical plumbagin.

From the cellular to tissue alterations induced by two rare earth elements in the mussel species Mytilus galloprovincialis: Comparison between exposure and recovery periods

The global effort to achieve carbon neutrality has led to an increased demand for renewable energy technologies and their raw materials, namely rare earth elements (REEs). These elements possess unique properties and are used in various applications. However, the increased use of REE-based technologies has resulted in higher amounts of electronic waste, leading to elevated REEs concentrations found in the aquatic environment, with poorly understood threats to wildlife. Praseodymium (Pr) and europium (Eu) are two REEs that, despite their potential environmental risks, have almost unknown effects on aquatic organisms. Therefore, the present study aimed to assess the impacts of different concentrations of Pr and Eu (0, 10, 20, 40, and 80 μg/L) in the mussel species Mytilus galloprovincialis, as well as their ability to recover from exposure to the highest concentration. Mussels accumulated both elements in a dose-dependent manner, with the accumulation of Pr being higher. Accompanying the increase of metabolism, mussels exposed to Pr not only enhanced the activity of the antioxidant enzymes superoxide dismutase (up to 40 μg/L) and glutathione reductase (at 80 μg/L) but also the activity of the biotransformation enzymes carboxylesterases (CbE's) and glutathione S-transferases (GSTs) (at 80 μg/L). Nevertheless, these defence mechanisms were not sufficient to prevent cellular damage. All the Eu concentrations induced cellular damage, despite an increase in the activity of biotransformation enzymes (CbE's and GSTs) in mussel tissue. According to the histopathology assessment, mussels were not able to recover after exposure to both elements and lower concentrations induced higher injuries in digestive tubules. This study highlights that exposure to Pr and Eu had adverse effects on M. galloprovincialis, even at the lowest tested concentration, which may eventually impact mussels' growth, reproductive capacity, and survival.

Conjugates of amiridine and thiouracil derivatives as effective inhibitors of butyrylcholinesterase with the potential to block β-amyloid aggregation.

New amiridine-thiouracil conjugates with different substituents in the pyrimidine fragment (R = CH3 , CF2 Н, CF3 , (CF2 )2 H) and different spacer lengths (n = 1-3) were synthesized. The conjugates rather weakly inhibit acetylcholinesterase (AChE) and exhibit high inhibitory activity (IC50 up to 0.752 ± 0.021 µM) and selectivity to butyrylcholinesterase (BChE), which increases with spacer elongation; the lead compounds are 11c, 12c, and 13c. The conjugates are mixed-type reversible inhibitors of both cholinesterases and practically do not inhibit the structurally related off-target enzyme carboxylesterase. The results of molecular docking to AChE and BChE are consistent with the experiment on enzyme inhibition and explain the structure-activity relationships, including the rather low anti-AChE activity and the high anti-BChE activity of long-chain conjugates. The lead compounds displace propidium from the AChE peripheral anion site (PAS) at the level of the reference compound donepezil, which agrees with the mixed-type mechanism of AChE inhibition and the main mode of binding of conjugates in the active site of AChE due to the interaction of the pyrimidine moiety with the PAS. This indicates the ability of the studied conjugates to block AChE-induced aggregation of β-amyloid, thereby exerting a disease-modifying effect. According to computer calculations, all synthesized conjugates have an ADME profile acceptable for drugs.

Computational analysis of carboxylesterase genes and proteins in non-pathogenic food bacterium Alicyclobacillus acidocaldarius: insights from proteogenomics.

Over recent years, Alicyclobacillus acidocaldarius, a Gram-positive nonpathogenic rod-shaped thermo-acid-tolerant bacterium, has posed numerous challenges for the fruit juice industry. However, the bacterium's unique characteristics, particularly its nonpathogenic and thermophilic capabilities, offer significant opportunities for genetic exploration by biotechnologists. This study presents the computational proteogenomics report on the carboxylesterase (CE) enzyme in A. acidocaldarius, shedding light on structural and evolutional of CEs from this bacterium. Our analysis revealed that the average molecular weight of CEs in A. acidocaldarius was 41kDa, with an isoelectric point around 5. The amino acid composition favored negative amino acids over positive ones. The aliphatic index and hydropathicity were approximately 88 and - 0.15, respectively. While the protein sequence showed no disulfide bonds in the CEs' structure, the presence of Cys amino acids was observed in the structure of CEs. Phylogenetic analysis presented more than 99% similarity between CEs, indicating their close evolutionary relationship. By applying homology modeling, the 3-dimensional structural models of the carboxylesterase were constructed, which with the help of structural conservation and solvent accessibility analysis highlighted key residues and regions responsible for enzyme stability and conformation. The specific patterns presented the total solvent accessibility of less than 25 (Å2) was in considerable position as well as Gly residues were noticeably have high accessibility to solvent in all structures. Ala was the more frequent amino acids in the conserved-SASA of carboxylesterases. Furthermore, unsupervised agglomerative hierarchical clustering based on solvent accessibility feature successfully clustered and even distinguished this enzyme from proteases from the same genome. These findings contribute to a deeper understanding of the nonpathogenic A. acidocaldarius carboxylesterase and its potential applications in biotechnology. Additionally, structural analysis of CEs would help to address potential solutions in fruit juice industry with utilization of computational structural biology.

Sublethal effects of chlorantraniliprole on biological characteristics, detoxifying enzyme activity and gene expression profile in the Allium mongolicum Regel leaf beetle Galeruca daurica (Coleoptera: Chrysomelidae)

AbstractAllium mongolicum Regel leaf beetle, Galeruca daurica (Coleoptera: Chrysomelidae) is rampantly harmful in Inner Mongolia grassland. However, the current management strategies for this pest still heavily rely on chemical control using traditional insecticides or those with novel action. In the study, we conducted an indoor bioassay to evaluate the sublethal effects of chlorantraniliprole on the biological characteristics. Additionally, we assessed the activity of detoxification enzymes, specifically the primary enzymes carboxylesterase (CarE) and cytochrome P450, and insect's secondary metabolic enzymes, namely glutathione S‐transferase (GST) and uridine diphosphate glycosyltransferase (UGT), as well as its gene expression profile. The developmental period of the 3rd instar larvae of G. daurica was significantly prolonged after treatment with chlorantraniliprole, which negatively affected the hatching, pupation and diapause rates as well as their body weight. The larvae showed different dynamics of the enzyme activities within 24 h of chlorantraniliprole treatment. Different sublethal concentrations of chlorantraniliprole showed an inducing effect on the activities, while no significant difference in activity was observed for P450. Expression profiling of detoxifying enzyme genes screened by transcriptome data has revealed that 6 CarE, 2 GST, 7 UGT and 31 P450 genes were up‐regulated. In conclusion, chlorantraniliprole's sublethal effect on 3rd instar G. daurica larvae resulted in a deceleration of their growth and developmental processes and significantly increased the activity levels of CarE, GST and UGT. The detoxifying proteins encoded by up‐regulation genes may involve in the detoxification metabolism of chlorantraniliprole. Our results provide a basis for understanding the molecular mechanisms of chlorantraniliprole action and detoxification in this key grassland insect pest.

Determination of Capecitabine and Its Metabolites in Plasma of Egyptian Colorectal Cancer Patients

The incidence of colorectal cancer (CRC) is increasing worldwide. It has variable signs and symptoms starting from changes in bowel habit to nausea and vomiting. Chemotherapeutic agents are often prescribed in CRC such as Capecitabine (CCB) and 5-Fluorouracil (FU). CCB is the prodrug of FU in oral dosage form, which makes it preferable by physicians, since no hospitalization is needed for drug administration. CCB is activated to FU in a three-step reaction producing 5′-deoxy-5-fluorocytidine (DFCR) (by carboxylesterase (CES) enzyme), then 5′-deoxy-5-fluorouridine (DFUR) (by cytidine deaminase (CDD) enzyme) and finally FU (by thymidine phosphorylase (TP) enzyme), the active form, which is later deactivated to give 5,6-dihydro-5-fluorouracil (DHFU). Different patients exhibit variable drug responses and adverse in response to CCB therapy, despite being treated by the same dose, which could be attributed to the occurrence of different possible enzyme single nucleotide polymorphisms (SNPs) along the activation and deactivation pathways of CCB. The most commonly occurring toxicities in CCB therapy are hand-foot syndrome and diarrhea. This study aims at developing and validating a new method for the simultaneous determination of CCB and its metabolites by HPLC-UV, followed by a correlation study with the toxicities occurring during therapy, where predictions of toxicity could be based on metabolites’ levels instead of the tedious process of genotyping. A new superior analytical method was optimized by a quality-by-design approach using DryLab® 2000 software achieving a baseline resolution of the six analytes within the least possible gradient time of 10 min. The method also showed linearity (in a range from 1 to 500 μg/mL), accuracy, precision and robustness upon validation: The LOD was found to be 3.0 ng/mL for DHFU and CCB, and 0.3 ng/mL for DFUR, DFCR and FU. The LOQ was found to be 10.0 ng/mL for DHFU and CCB, and 1.0 ng/mL for DFUR, DFCR and FU. The clinical results showed a positive correlation between the concentration of DFCR and mucositis and between the concentration of DFUR and hand-foot syndrome, confirming that this technique could be used for predicting such toxicities.

Investigating pH Effects on Enzymes Catalyzing Polysorbate Degradation by Activity-Based Protein Profiling

Host cell proteins (HCPs) are process-related impurities that can negatively impact the quality of biotherapeutics. Some HCPs possess enzymatic activity and can affect the active pharmaceutical ingredient (API) or excipients such as polysorbates (PS). PSs are a class of non-ionic surfactants commonly used as excipients in biotherapeutics to enhance the stability of APIs. The enzyme activity of certain HCPs can result in the degradation of PSs, leading to particle formation and decreased shelf life of biotherapeutics. Identifying and characterizing these HCPs is therefore crucial. This study employed the Activity-Based Protein Profiling (ABPP) technique to investigate the effect of pH on the activity of HCPs that have the potential to degrade polysorbates. Two probes were utilized: the commercially available fluorophosphonate (FP)-Desthiobiotin probe and a probe based on the antiobesity drug, Orlistat. Over 50 HCPs were identified, showing a strong dependence on pH-milieu regarding their enzyme activity. These findings underscore the importance of accounting for pH variations in the ABPP method and other investigations of HCP activity. Notably, the Orlistat-based probe (OBP) enabled us to investigate the enzymatic activity of a wider range of HCPs, emphasizing the advantage of using more than one probe for ABPP. Finally, this study led to the discovery of previously unreported active enzymes, including three HCPs from the carboxylesterase enzyme family.

Carboxylesterase from edible legume seeds and their sensitivity to organophosphate and carbamate pesticides

Carboxylesterase from edible legume seeds, including black gram bean (Vigna mungo (L.) Hepper), mung bean (Vigna radiata (L.) Wilczek) and red bean (Vigna umbellata (Thunb.) Ohwi and Ohashi) has been partially purified using the ammonium sulphate salting-out procedure. The specific activity of the partially purified carboxylesterase extracted from black gram bean, mung bean and red bean are 3.96±0.26, 1.51±0.10 and 1.29±0.08 U/mg, respectively (p<0.05). The carboxylesterase from black gram bean showed very high sensitivity to 24 types of organophosphate pesticides (OPs) and 7 types of carbamate pesticides (CAs) at the concentrations of 0.001-3.0 µg/ml. The limit of detection (LOD) obtained for OPs and CAs pesticide residues were 0.002 and 0.01 mg/kg, respectively. For performance assessment of methods for pesticide determination in vegetables, all performance parameter values, including sensitivity, accuracy, and specificity of the carboxylesterase enzyme inhibition method, are 100% compared with commercial pesticide test kits and are 70.5%, 90%, and 100%, respectively, compared with chromatographic method analysis.