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Carboxylesterases comprise a major class of α/β‐fold hydrolases responsible for the cleavage and formation of ester bonds. Found ubiquitously in nature, these enzymes are crucial for the metabolism of both endogenous and exogenous carboxyl esters in animals, plants and microorganisms. Beyond their essential physiological roles, carboxylesterases stand out as one of the important classes of biocatalysts for biotechnology. BlEst2, an enzyme previously classified as Bacillus licheniformis esterase, remains largely uncharacterized. In the present study, we elucidate the structural biology, molecular dynamics and biochemical features of BlEst2. Our findings reveal a canonical α/β‐hydrolase fold similar to the ESTHER block L of lipases, further augmented by two additional accessory C‐terminal domains. Notably, the catalytic domain demonstrates two insertions, which occupy conserved locations in α/β‐hydrolase proteins and commonly form the lid domain in lipase structures. Intriguingly, our in vitro cleavage of C‐terminal domains revealed the structure of the active form of BlEst2. Upon activation, BlEst2 showed a markedly elevated hydrolytic activity. This observation implies that the intramolecular C‐terminal domain serves as a regulatory intramolecular inhibitor. Interestingly, despite exhibiting esterase‐like activity, BlEst2 structural characteristics align more closely with lipases. This suggests that BlEst2 could potentially represent a previously unrecognized subgroup within the realm of carboxyl ester hydrolases. [ABSTRACT FROM AUTHOR]

Carboxylesterases serve as potent biocatalysts in the enantioselective synthesis of chiral carboxylic acids and esters. However, naturally occurring carboxylesterases exhibit limited enantioselectivity, particularly toward ethyl 3-cyclohexene-1-carboxylate (CHCE, S1), due to its nearly symmetric structure. While machine learning effectively expedites directed evolution, the lack of models for predicting the enantioselectivity for carboxylesterases has hindered progress, primarily due to challenges in obtaining high-quality training datasets. In this study, we devise a high-throughput method by coupling alcohol dehydrogenase to determine the apparent enantioselectivity of the carboxylesterase AcEst1 from Acinetobacter sp. JNU9335, generating a high-quality dataset. Leveraging seven features derived from biochemical considerations, we quantitively describe the steric, hydrophobic, hydrophilic, electrostatic, hydrogen bonding, and π-π interaction effects of residues within AcEst1. A robust gradient boosting regression tree model is trained to facilitate stereodivergent evolution, resulting in the enhanced enantioselectivity of AcEst1 toward S1. Through this approach, we successfully obtain two stereocomplementary variants, DR3 and DS6, demonstrating significantly increased and reversed enantioselectivity. Notably, DR3 and DS6 exhibit utility in the enantioselective hydrolysis of various symmetric esters. Comprehensive kinetic parameter analysis, molecular dynamics simulations, and QM/MM calculations offer insights into the kinetic and thermodynamic features underlying the manipulated enantioselectivity of DR3 and DS6. Carboxylesterases catalyze the enantioselective synthesis of chiral carboxylic acids and esters but exhibit limited enantioselectivity, particularly toward ethyl 3-cyclohexene-1- carboxylate (CHCE). Here, the authors establish a high-throughput method to generate a high-quality dataset for carboxylesterase AcEst1 from Acinetobacter sp. JNU9335 using CHCE as the substrate, use it to train a machine learning predictor and design two stereocomplementary mutants to synthesize both enantiomers of chiral CHCE. [ABSTRACT FROM AUTHOR]

Secondary metabolites are bioactive compounds believed to contribute to the pharmacological properties of plants. MicroRNAs (miRNAs) are small non-coding RNA molecules involved in post-transcriptional regulation and are thought to play an important role in regulating secondary metabolism biosynthesis. Nevertheless, the extent of miRNA involvement in secondary metabolism remains minimal. Nigella sativa (black cumin/black seed) is a popular medicinal and culinary plant known for its pharmaceutical properties; however, its genomic information is scarce. In this study, next-generation sequencing (NGS) technology was employed to obtain the miRNA profile of N. sativa, and their involvement in secondary metabolite biosynthesis was explored. A total of 25,139,003 unique reads ranging from 16 to 40 nucleotides were attained, out of which 240 conserved and 34 novel miRNAs were identified. Moreover, 6083 potential target genes were recognized in this study. Several conserved and novel black cumin miRNAs were found to target enzymes involved in the terpenoid, diterpenoid, phenylpropanoid, carotenoid, flavonoid, steroid, and ubiquinone biosynthetic pathways, among others, for example, beta-carotene 3-hydroxylase, gibberellin 3 beta-dioxygenase, trimethyltridecatetraene synthase, carboxylic ester hydrolases, acetyl-CoA C-acetyltransferase, isoprene synthase, peroxidase, shikimate O-hydroxycinnamoyltransferase, etc. Furthermore, sequencing data were validated through qPCR by checking the relative expression of eleven randomly selected conserved and novel miRNAs (nsa-miR164d, nsa-miR166a, nsa-miR167b, nsa-miR171a, nsa-miR390b, nsa-miR396, nsa-miR159a, nsa-miRN1, nsa-miRN29, nsa-miRN32, and nsa-miRN34) and their expression patterns were found to be corroborated with the sequencing data. We anticipate that this work will assist in clarifying the implications of miRNAs in plant secondary metabolism and aid in the generation of artificial miRNA-based strategies to overproduce highly valuable secondary metabolites from N. sativa. [ABSTRACT FROM AUTHOR]

Pig carboxylesterases (CESs) are recognized as enzymes that metabolize xenobiotics, and many drugs used in pig breeding that contain ester groups can be hydrolyzed by CESs. However, there are a limited number of publications focused on the molecular weight, inhibitor effects, and enzyme kinetics of porcine cytosolic carboxylesterase. This study presents the first investigation of the presence, kinetics, inhibition, and amino acid sequence of CES1, the major cytosolic CES isozyme in pig liver. The molecular weight of pig liver cytosolic CES1 was estimated to be approximately 179 kDa, and the amino acid sequence of the enzyme subunit was determined. The inhibition of CES1 by the antiviral drugs remdesivir and sofosbuvir was studied, and the IC50 values were determined. The results indicate that remdesivir and sofosbuvir can act as potent modulators of CES1. The inhibitors exhibited mixed‐type inhibition on pig liver cytosol CES1. It was found that the affinity of the remdesivir and sofosbuvir for free enzyme is greater than that for the enzyme‐substrate complex. A spectrofluorimetric method confirmed the inhibition findings, as the fluorescence intensity of remdesivir decreased upon interaction with the enzyme. [ABSTRACT FROM AUTHOR]

Colorado potato beetle (CPB) is an extraordinary example of pest resistance to insecticides. It is proved that increased activity of CPB's ALiE / carboxylesterase is closely related to resistance to organophosphate insecticides. ALiE activity of different populations was tested, using spectrophotometry. The frequencies of ALiE activity of individual larvae were similar to the binomial distribution. For more resistant populations to organophosphates, the whole graph is shifted to the area with higher enzyme activity. Consequently, individuals with lower ALiE activity disappear from the population while individuals with higher activity appear more frequently. The analysis of single larvae ALiE activity showed a fairly high homogeneity of the examined populations, except for the population Kaona. Examination of single insect ALiE activity is viable and provides insight into the population, which is important for further genetic testing. [ABSTRACT FROM AUTHOR]

In the human body, carboxylesterases (CEs) play crucial roles in xenobiotic metabolism and lipid homeostasis. But abnormal expression of CEs is highly associated with some diseases, such as hyperlipidemia, diabetes, and liver cancer. Therefore, it is of great importance to develop an efficient tool for the accurate detection of CEs in living organisms. Herein, an innovative near-infrared (NIR) fluorescent probe, TTAP−AB, was designed for CE detection based on the aggregation-induced emission (AIE) mechanism. This probe exhibits rapid response (2 min), excellent sensitivity (limit of detection = 8.14 × 10−6 U/mL), and high selectivity to CEs. Additionally, owing to its good biocompatibility, the TTAP−AB probe enables the monitoring of dynamic changes in CE levels under drug-induced modulation in living cells and zebrafish. More importantly, the TTAP−AB probe was successfully employed to image liver tumors and assist in tumor resection through the real-time monitoring of CEs, indicating that TTAP−AB is promising to guide liver cancer surgery. Therefore, the TTAP−AB probe can not only enrich the strategies for CE detection in biological systems but also has great potential for some clinical imaging applications, including medical diagnosis, preclinical research, and imaging-guided surgery. [ABSTRACT FROM AUTHOR]

Homocysteine thiolactone (HTL), a toxic metabolite of homocysteine (Hcy) in hyperhomocysteinemia (HHcy), is known to modify protein structure and function, leading to protein damage through formation of N‐Hcy‐protein. HTL has been highly linked to HHcy‐associated cardiovascular and neurodegenerative diseases. The protective role of HTL hydrolases against HTL‐associated vascular toxicity and neurotoxicity have been reported. Although several endogeneous enzymes capable of hydrolyzing HTL have been identified, the primary enzyme responsible for its metabolism remains unclear. In this study, three human carboxylesterases were screened to explore new HTL hydrolase and human carboxylesterase 1 (hCES1) demonstrates the highest catalytic activity against HTL. Given the abundance of hCES1 in the liver and the clinical significance of its single‐nucleotide polymorphisms (SNPs), six common hCES1 nonsynonymous coding SNP (nsSNPs) variants were examined and characterized for their kinetic parameters. Variants E220G and G143E displayed 7.3‐fold and 13.2‐fold lower catalytic activities than its wild‐type counterpart. In addition, the detailed catalytic mechanism of hCES1 for HTL hydrolysis was computational investigated and elucidated by Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) method. The function of residues E220 and G143 in sustaining its hydrolytic activity of hCES1 was analyzed, and the calculated energy difference aligns well with experimental‐derived results, supporting the validity of our computational insights. These findings provide insights into the potential protective role of hCES1 against HTL‐associated toxicity, and warrant future studies on the possible association between specific genetic variants of hCES1 with impaired catalytic function and clinical susceptibility of HTL‐associated cardiovascular and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

[2,3-diamino-N-(4-(benzo[d]thiazol-2-yl)phenyl)propanamide], named as ETN101, is a novel therapeutic agent for hepatocellular carcinoma. In vitro studies examined ETN101 metabolites in human, mouse, rat, dog, and monkey hepatocytes and identified the drug-metabolizing enzymes involved using cDNA-expressed human recombinant cytochrome P450s (CYPs), carboxylesterases (CESs), N-acetyltransferase (NAT) 1, and human liver cytosol. ETN101 showed similar metabolic stability across hepatocytes from five species, with particularly comparable stability in humans, rats, and monkeys. Its half-life was 75.0 min in humans, 68.9 in rats, 73.1 in monkeys, 120.4 in mice, and 112.7 in dogs. Thirty-four ETN101 metabolites, including the major metabolite M1, were identified using liquid chromatography–high-resolution mass spectrometry. ETN101 was primarily metabolized to M1 and CYP1A2 is exclusively responsible for M1 metabolism. Both NAT1 and NAT2 were responsible for the N-acetylation of M1 to M2. ETN101 remained stable in human CESs. In conclusion, this study provides comprehensive insights into the metabolic characteristics of ETN101, valuable for its toxicological and clinical development. [ABSTRACT FROM AUTHOR]

Discharges to the aquatic environment of pharmaceuticals represent a hazard to the aquatic organisms. Subchronic assay with 17‐alpha‐ethinylestradiol (EE2) and in vitro essays with pharmaceuticals of environmental concern were conducted to examine the sensitivity of tissue acetylcholinesterase (AChE) and carboxylesterase (CbE) activities of Tinca tinca to them. Subchronic exposure to 17‐alpha‐EE2 caused significant effects on brain, liver, and muscle CbE, but no on AChE activities. Most of the pharmaceuticals tested in vitro were considered as weak inhibitors of tissular AChE activity. Depending on the tissues, some compounds were classified as moderate inhibitors of CbE activity while other were categorized as weak enzymatic inhibitors. An opposite trend was observed depending on the tissue, while brain and liver CbE activities were inhibited, the muscle CbE activity was induced. Changes experienced on enzymatic activities after exposure to pharmaceuticals might affect the physiological functions in which these enzymes are involved. In vitro exposure to 17‐alpha‐EE2 in tench could be an informative, but not a surrogate model to know the effect of this synthetic estrogen on AChE and CbE activities. [ABSTRACT FROM AUTHOR]

Carboxylic ester hydrolases with the capacity to degrade polyesters are currently highly sought after for their potential use in the biological degradation of PET and other chemically synthesized polymers. Here, we describe MarCE, a carboxylesterase family protein identified via genome mining of a Maribacter sp. isolate from the marine sponge Stelligera stuposa. Based on phylogenetic analysis, MarCE and its closest relatives belong to marine‐associated genera from the Cytophaga–Flavobacterium–Bacteroides taxonomic group and appear evolutionarily distinct to any homologous carboxylesterases that have been studied to date in terms of structure or function. Molecular docking revealed putative binding of BHET, a short‐chain PET derivative, onto the predicted MarCE three‐dimensional structure. The synthetic ester‐degrading activity of MarCE was subsequently confirmed by MarCE‐mediated hydrolysis of 2 mM BHET substrate, indicated by the release of its breakdown products MHET and TPA, which were measured, respectively, as 1.28 and 0.12 mM following 2‐h incubation at 30°C. The findings of this study provide further insight into marine carboxylic ester hydrolases, which have the potential to display unique functional plasticity resulting from their adaptation to complex and fluctuating marine environmentsw. [ABSTRACT FROM AUTHOR]

Two new copper(II) complexes (1 and 2) with reduced L -histidine Schiff bases were synthesized and characterized using single-crystal X-ray diffraction, powder X-ray diffraction, UV-visible spectroscopy, and thermal analysis. Single-crystal analysis reveals that 5-methyl-containing 1 possesses a simple phenoxo-bridged binuclear unit, while 5-bromine-substituted 2 possesses a metal–organic cage (MOC) structure. The as-prepared complexes as artificial metallohydrolases displayed catalytic activity towards the hydrolysis of both PNPP (p-nitrophenyl picolinate) and PNPA (p-nitrophenyl acetate). These two complexes provided 28-fold (for 1) and 39-fold (for 2) rate enhancements in comparison with spontaneous hydrolysis of PNPA, respectively. With regard to PNPP, its hydrolysis was accelerated by two orders of magnitude in the presence of 1 or 2. Additionally, positive micellar effects of bis(hexadecyldimethylammonium)hexane bromide (16-6-16) and N-octyldocosylammonium bromide (C22H45N(CH3)2C8H17Br, abbr. C22/8) were observed for the PNPA hydrolysis by 1 or 2, resulting in 1.3 to 3.9 times acceleration compared to that in buffered aqueous solution. In micellar solution of N,N-dimethyl-n-lauroylsarcosine sodium (LSS), no obvious rate enhancement was observed for the PNPA hydrolysis by 1 or 2. [ABSTRACT FROM AUTHOR]

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Pollution of water resources with pesticides negatively affects aquatic organisms and makes water bodies unsuitable for use by humans. Chemical analysis methods do not provide information about the impact of a detected substance and its individual components on the ecosystem. The article presents an analysis and synthesis of published data on the possibilities and features of the use of aquatic enzymes as biomarkers of pollution of aquatic ecosystems with pesticides. Publications over the past 20 years, indexed in the PubMed, Crossref, Web of Science, Scopus, and RSCI databases devoted to the issues of pesticide pollution of water bodies and the use of enzymes, in particular nonspecific esterases, in biomonitoring were analyzed. The concept of "biomarker" is revealed, groups of biomarkers, the purposes of their use, advantages and disadvantages as a source of information about the state of the ecosystem are listed. Particular attention is paid to the study of nonspecific esterases in aquatic organisms (fish, mollusks, crustaceans, and amphibians). The main types of substrates used to measure the activity of esterase isoforms and the features of changes in enzymatic activity in response to exposure to pesticides from different chemical groups (organophosphorus compounds, carbamates, and pyrethroids) are considered. The factors influencing the activity of nonspecific esterases of aquatic organisms and limiting their use for assessing pollution of aquatic ecosystems are identified. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the most commonly used mammalian cell line for the production of complex therapeutic glycoproteins. As CHO cells have evolved as part of a multicellular organism, they harbor many cellular functions irrelevant for their application as production hosts in industrial bioprocesses. Consequently, CHO cells have been the target for numerous genetic engineering efforts in the past, but a tailored host cell chassis holistically optimized for its specific task in a bioreactor is still missing. While the concept of genome reduction has already been successfully applied to bacterial production cells, attempts to create higher eukaryotic production hosts exhibiting reduced genomes have not been reported yet. Here, we present the establishment and application of a large-scale genome deletion strategy for targeted excision of large genomic regions in CHO cells. We demonstrate the feasibility of genome reduction in CHO cells using optimized CRISPR/Cas9 based experimental protocols targeting large non-essential genomic regions with high efficiency. Achieved genome deletions of non-essential genetic regions did not introduce negative effects on bioprocess relevant parameters, although we conducted the largest reported genomic excision of 864 kilobase pairs in CHO cells so far. The concept presented serves as a directive to accelerate the development of a significantly genome-reduced CHO host cell chassis paving the way for a next generation of CHO cell factories. • Establishment and application of large-scale genome excision in CHO cells. • Targeting of non-essential genomic regions via CRISPR/Cas9 with high efficiency. • Achievement of the largest reported genomic excision of 864 kilobase pairs in CHO cells. • No negative effects on bioprocess relevant parameters after gene excision. • The concept serves as a directive towards a significantly genome reduced CHO host cell chassis. [ABSTRACT FROM AUTHOR]

Pro-drugs, which ideally release their active compound only at the site of action, i.e., in a cancer cell, are a promising approach towards an increased specificity and hence reduced side effects in chemotherapy. A popular form of pro-drugs is esters, which are activated upon their hydrolysis. Since carboxylesterases that catalyse such a hydrolysis reaction are also abundant in normal tissue, it is of great interest whether a putative pro-drug is a probable substrate of such an enzyme and hence bears the danger of being activated not just in the target environment, i.e., in cancer cells. In this work, we study the binding mode of carboxylesters of the drug molecule camptothecin, which is an inhibitor of topoisomerase I, of varying size to human carboxylesterase 2 (HCE2) by molecular docking and molecular dynamics simulations. A comparison to irinotecan, known to be a substrate of HCE2, shows that all three pro-drugs analysed in this work can bind to the HCE2 protein, but not in a pose that is well suited for subsequent hydrolysis. Our data suggest, moreover, that for the irinotecan substrate, a reactant-competent pose is stabilised once the initial proton transfer from the putative nucleophile Ser202 to the His431 of the catalytic triad has already occurred. Our simulation work also shows that it is important to go beyond the static models obtained from molecular docking and include the flexibility of enzyme–ligand complexes in solvents and at a finite temperature. Under such conditions, the pro-drugs studied in this work are unlikely to be hydrolysed by the HCE2 enzyme, indicating a low risk of undesired drug release in normal tissue. [ABSTRACT FROM AUTHOR]

Fumonisins (FBs), particularly fumonisin B1 (FB1) and fumonisin B2 (FB2) produced mainly by Fusarium verticillioide and Fusarium proliferatum, are common contaminants in animal feed and pose a serious threat to both animal and human health. The use of microbial enzymes to efficiently and specifically convert fumonisins into non-toxic or low-toxic metabolites has emerged as the most promising approach. However, most of the available enzymes have only been evaluated in vitro and lack systematic evaluation in vivo. In this study, the detoxification efficacy of two carboxylesterases, FumD (FUMzyme®) and FumDSB, was evaluated comparatively in piglets. The results show that feeding piglets 4.4 mg/kg FBs-contaminated diets for 32 days did not significantly affect the average daily gain, organ indices, and immunoglobulins of the piglets. However, a significant reduction (21.2%) in anti-inflammatory cytokine interleukin-4 was observed in the FBs group, and supplementation with FUMzyme® and FumDSB significantly increased interleukin-4 by 62.1% and 28.0%, respectively. In addition, FBs-contaminated diets resulted in a 3-fold increase in the serum sphinganine/sphingosine (Sa/So) ratio, which is a specific biomarker that has been used to accurately reflect fumonisin levels. The serum Sa/So ratio was significantly reduced by 48.8% after the addition of FUMzyme®, and was insignificantly reduced by 8.2% in the FumDSB group. These results suggested that FUMzyme was more effective than FumDSB in mitigating FBs toxicity in piglets by down-regulating the Sa/So ratio. [ABSTRACT FROM AUTHOR]

1. Carboxylesterase (CES) has been studied extensively, mostly with substrates in the monoester structures. We investigated the relationship between indomethacin diester prodrugs and metabolic activation by microsomes and recombinant human CES. 2. Eight indomethacin diester prodrugs were synthesised in two steps. They were used as substrates and hydrolysis rates were calculated. 3. As a result, the major hydrolysis enzyme was CES. The hydrolysis rate of recombinant CES2A1 was comparable to that of recombinant CES1A1. 4. In this study, by changing the structure of the prodrug to a diester structure, it was found that CES2 activity was equivalent to CES1 activity. 5. It should be noted that the use of diester prodrugs in prodrug discovery, where organ-specific hydrolysis reactions are expected, may not yield the expected results. [ABSTRACT FROM AUTHOR]

In the human body, carboxylesterases (CEs) play crucial roles in xenobiotic metabolism and lipid homeostasis. But abnormal expression of CEs is highly associated with some diseases, such as hyperlipidemia, diabetes, and liver cancer. Therefore, it is of great importance to develop an efficient tool for the accurate detection of CEs in living organisms. Herein, an innovative near-infrared (NIR) fluorescent probe, TTAP−AB, was designed for CE detection based on the aggregation-induced emission (AIE) mechanism. This probe exhibits rapid response (2 min), excellent sensitivity (limit of detection = 8.14 × 10−6 U/mL), and high selectivity to CEs. Additionally, owing to its good biocompatibility, the TTAP−AB probe enables the monitoring of dynamic changes in CE levels under drug-induced modulation in living cells and zebrafish. More importantly, the TTAP−AB probe was successfully employed to image liver tumors and assist in tumor resection through the real-time monitoring of CEs, indicating that TTAP−AB is promising to guide liver cancer surgery. Therefore, the TTAP−AB probe can not only enrich the strategies for CE detection in biological systems but also has great potential for some clinical imaging applications, including medical diagnosis, preclinical research, and imaging-guided surgery.

Moth sex pheromones are a classical model for studying sexual selection. Females typically produce a species-specific pheromone blend that attracts males. Revealing the enzymes involved in the interspecific variation in blend composition is key for understanding the evolution of these sexual communication systems. The nature of the enzymes involved in the variation of acetate esters, which are prominent compounds in moth pheromone blends, remains unclear. We identify enzymes involved in acetate degradation using two closely related moth species: Heliothis (Chloridea) subflexa and H. (C.) virescens, which have different quantities of acetate esters in their sex pheromone. Through comparative transcriptomic analyses and CRISPR/Cas9 knockouts, we show that two lipases and two esterases from H. virescens reduce the levels of pheromone acetate esters when expressed in H. subflexa females. Together, our results show that lipases and carboxylesterases are involved in tuning Lepidoptera pheromones composition. Moth sex pheromones, pivotal for mate attraction, are a classic model for sexual selection. Through introgression, transcriptomics and knocking out genes, this study identifies lipases and esterases that affect pheromone blend composition, offering insights into moth pheromone evolution. [ABSTRACT FROM AUTHOR]

Key message: The transcript level of alcohol acyltransferase 1 (AAT1) may be the main factor influencing the variations in volatile esters that characterizing the fruity/exotic aroma of pepper fruit. Volatile esters are key components for characterizing the fruity/exotic aroma of pepper (Capsicum spp.) fruit. In general, the volatile ester content in the fruit is the consequence of a delicate balance between their synthesis by alcohol acyltransferases (AATs) and degradation by carboxylesterases (CXEs). However, the precise role of these families of enzymes with regard to volatile ester content remains unexplored in Capsicum. In this study, we found that the volatile ester content was relatively low in C. annuum and much higher in C. chinense, particularly in pungent varieties. Additionally, fruits collected from multiple non-pungent C. chinense varieties, which harbor loss-of-function mutations in capsaicinoid biosynthetic genes, acyltransferase (Pun1), putative aminotransferase (pAMT), or putative ketoacyl-ACP reductase (CaKR1) were analyzed. The volatile ester contents of non-pungent C. chinense varieties (pamt/pamt) were equivalent to those of pungent varieties, but their levels were significantly lower in non-pungent NMCA30036 (pun12/pun12) and C. chinense (Cakr1/Cakr1) varieties. Multiple AAT-like sequences were identified from the pepper genome sequences, whereas only one CXE-like sequence was identified. Among these, AAT1, AAT2, and CXE1 were isolated from fruits of C. chinense and C. annuum. Gene expression analysis revealed that the AAT1 transcript level is a potential determinant of fruit volatile ester variations in Capsicum. Furthermore, enzymatic assays demonstrated that AAT1 is responsible for the biosynthesis of volatile esters in pepper fruit. Identification of a key gene for aroma biosynthesis in pepper fruit will provide a theoretical basis for the development of molecular tools for flavor improvement. [ABSTRACT FROM AUTHOR]

Background and objective: Side effects of irinotecan treatment can be dose limiting and may impair quality of life. In this study, we investigated the correlation between single nucleotide polymorphisms (SNPs) in genes encoding enzymes involved in the irinotecan metabolism and transport, outside UGT1A1, and irinotecan-related toxicity. We focused on carboxylesterases, which are involved in formation of the active metabolite SN-38 and on drug transporters. Methods: Patients who provided written informed consent at the Erasmus Medical Center Cancer Institute to the Code Geno study (local protocol: MEC02-1002) or the IRI28-study (NTR-6612) were enrolled in the study and were genotyped for 15 SNPs in the genes CES1, CES2, SLCO1B1, ABCB1, ABCC2, and ABCG2. Results: From 299 evaluable patients, 86 patients (28.8%) developed severe irinotecan-related toxicity. A significantly higher risk of toxicity was seen in ABCG2 c.421C>A variant allele carriers (P = 0.030, OR 1.88, 95% CI 1.06–3.34). Higher age was associated with all grade diarrhea (P = 0.041, OR 1.03, 95% CI 1.00–1.06). In addition, CES1 c.1165-41C>T and CES1 n.95346T>C variant allele carriers had a lower risk of all-grade thrombocytopenia (P = 0.024, OR 0.42, 95% CI 0.20–0.90 and P = 0.018, OR 0.23, 95% CI 0.08–0.79, respectively). Conclusion: Our study indicates that ABCG2 and CES1 SNPs might be used as predictive markers for irinotecan-induced toxicity. [ABSTRACT FROM AUTHOR]

cis-Diamminedichloroplatinum (CDDP) is widely used for the treatment of various solid cancers. Here we reported that CDDP increased the expression and enzymatic activities of carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2), along with the upregulation of pregnane X receptor (PXR) and the downregulation of differentiated embryonic chondrocyte-expressed gene 1 (DEC1) in human hepatoma cells, primary mouse hepatocytes, mouse liver and intestine. The overexpression or knockdown of PXR alone upregulated or downregulated the CES1 and CES2 expression, respectively. The increases in CES1 and CES2 expression levels induced by CDDP abolished or enhanced by PXR knockdown or overexpression, implying that CDDP induces carboxylesterases through the activation of PXR. Likewise, the overexpression or knockdown of DEC1 alone significantly decreased or increased PXR and its targets. Moreover, the increases of PXR and its targets induced by CDDP were abolished or alleviated by the overexpression or knockdown of DEC1. The overexpression or knockdown of DEC1 affected the response of PXR to CDDP, but not vice versa, suggesting that CDDP increases carboxylesterases by upregulating PXR mediated by the decrease of DEC1. In addition, CDDP did not increase DEC1 mRNA degradation but suppressed DEC1 promoter reporter activity, indicating that it suppresses DEC1 transcriptionally. The combined use of CDDP and irinotecan had a synergistic effect on two cell lines, especially when CDDP was used first. [ABSTRACT FROM AUTHOR]

Russian wheat aphid, Diuraphis noxia is one of the economic and polyphagous pests of cereals that are exposed to various chemicals such as phenols. In this research, to investigate the possible effect of host phenolic content on the effectiveness of chemical control, Imidacloprid susceptibility was detected for aphids reared on wheat (Shiroodi and Gascogne) and triticale (Sanabad and Juanillo 92) cultivars, under greenhouse bioassay. The LC50 values of Imidacloprid against the mentioned populations were 2.41, 3.56, 4.89, and 5.73 mg a.i. L-1, respectively. Therefore, the highest Imidacloprid susceptibility belonged to the Shiroodi population. Based on biochemical studies, the activity of alpha and beta esterases, glutathione S transferases, and cytochrome P450 for the Gascogne population was 1.32, 1.16, 1.16, and 1.5 times that of the Shiroodi population, respectively. These ratios were 1.82, 1.96, 1.4, and 2 for the Sanabad population and 2.09, 2.48, 1.51, and 2.25 for the Juanillo 92 population, respectively. Moreover, the phenol content of the mentioned cultivars was 2.19, 2.71, 3.48, and 3.87 mg per gram of leaf, respectively. The data analysis showed a positive and significant correlation between the cereals' phenolic content with the research subjects. Considering the biochemical relationships between D. noxia and its host plants, the value of plant phenolics can be suggested as a suitable index for the prediction of aphid response to Imidacloprid. [ABSTRACT FROM AUTHOR]

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CXEs) catalyze the hydrolysis of carboxylic acid esters into the corresponding acids and alcohols. Previously, a carboxylesterase gene was isolated from pepper (Capsicum annuum L.) and designated as PepEST. This gene is highly expressed during the incompatible interaction between ripe fruits of pepper and an anthracnose fungus (Colletotrichum gloeosporioides), and the PepEST protein exhibits an antifungal activity by suppressing fungal growth with the inhibition of appressorium formation. In this study, a PepEST-homologous gene was newly isolated from the pepper genome, which is designated as PepEST2 (the original PepEST gene is re-named here as PepEST1). Compared with PepEST1 whose expression is only induced by inoculation of the fungus, PepEST2 is not induced by fungus infection, rather expressed mainly in roots. Subcellular localization analysis using tobacco protoplasts showed that both PepEST1 and PepEST2 proteins are localized in the cytoplasm, and enzymatic activity assays showed similar Vmax and Km values of PepEST2 to those of PepEST1. Antifungal activity assays against C. gloeosporioides demonstrated that not only PepEST1 but also PepEST2 was able to suppress the fungal growth by inhibiting the appressorium formation. In addition, the antifungal activity of PepEST2 was higher than that of PepEST1. Collectively, this study suggests that two closely related members of pepper CXEs are functionally diversified due to the differential expression patterns, and also provides PepEST2 as an alternative gene to confer disease resistance to plants. [ABSTRACT FROM AUTHOR]

Insect carboxylesterases (CXEs) can be expressed in multiple tissues and play crucial roles in detoxifying xenobiotic insecticides and degrading olfactory cues. Therefore, they have been considered as an important target for development of eco-friendly insect pest management strategies. Despite extensive investigation in most insect species, limited information on CXEs in sibling moth species is currently available. The Ectropis obliqua Prout and Ectropis grisescens Warren are two closely related tea geometrid species, which share the same host of tea plant but differ in geographical distribution, sex pheromone composition, and symbiotic bacteria abundance, providing an excellent mode species for studies of functional diversity of orthologous CXEs. In this study, we focused on EoblCXE14 due to its previously reported non-chemosensory organs-biased expression. First, the EoblCXE14 orthologous gene EgriCXE14 was cloned and sequence characteristics analysis showed that they share a conserved motif and phylogenetic relationship. Quantitative real-time polymerase chain reaction (qRT-PCR) was then used to compare the expression profiles between two Ectropis spp. The results showed that EoblCXE14 was predominately expressed in E. obliqua larvae, whereas EgriCXE14 was abundant in E. grisescens at multiple developmental stages. Interestingly, both orthologous CXEs were highly expressed in larval midgut, but the expression level of EoblCXE14 in E. obliqua midgut was significantly higher than that of EgriCXE14 in E. grisescens midgut. In addition, the potential effect of symbiotic bacteria Wolbachia on the CXE14 was examined. This study is the first to provide comparative expression profiles of orthologous CXE genes in two sibling geometrid moth species and the results will help further elucidate CXEs functions and identify a potential target for tea geometrid pest control. [ABSTRACT FROM AUTHOR]

An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0–10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure–function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields. [ABSTRACT FROM AUTHOR]

Carboxylesterases (CEs) play important physiological roles in the human body and are involved in numerous cellular processes. Monitoring CEs activity has great potential for the rapid diagnosis of malignant tumors and multiple diseases. Herein, we developed a new phenazine-based "turn-on" fluorescent probe DBPpys by introducing 4-bromomethyl-phenyl acetate to DBPpy, which can selectively detect CEs with a low detection limit (9.38 × 10−5 U/mL) and a large Stokes shift (more than 250 nm) in vitro. In addition, DBPpys can also be converted into DBPpy by carboxylesterase in HeLa cells and localized in lipid droplets (LDs), emitting bright near-infrared fluorescence under the irradiation of white light. Moreover, we achieved the detection of cell health status by measuring the intensity of NIR fluorescence after co-incubation of DBPpys with H2O2-pretreated HeLa cells, indicating that DBPpys has great potential applications for assessing CEs activity and cellular health. [ABSTRACT FROM AUTHOR]

Abamectin is an important and environmentally friendly antibiotic pesticide, and its resistance has become an area of intense research. In this study, we employed laboratory bioassays, biochemical analyses, and life history analyses to systematically evaluate abamectin resistance, the underlying biochemical mechanisms of resistance, and the reproductive and developmental characteristics of the arthropod pest Bryobia praetiosa. An abamectin-resistant B. praetiosa strain (RR) was obtained by intensive selection of an abamectin-susceptible strain (SS) using abamectin exposure in the laboratory for 24 generations, yielding a resistance index (RI) of 30.11. Comparison of detoxification enzymes suggested that the specific activities of carboxylesterases (CarEs), glutathione S-transferases (GSTs), and mixed-function oxidases (MFOs) in the RR strain were higher than those of the SS strain by 1.13-, 1.75-, and 4.02-fold, respectively. GST and MFOS protein concentrations were also significantly different between RR and SS strains. Further analysis of life history parameters demonstrated that the developmental duration and lifespan of RR strain in addition to egg numbers were markedly decreased relative to SS strain. The net reproductive rate (R0), finite rate of increase (λ), and population doubling time (Dt) of RR strain were significantly lower than those of SS strain. The results indicated that the RR strain was more disadvantageous in developmental and reproductive characteristics than the SS strain. In addition, the relative fitness value of the resistant strain is about half (Rf = 0.49) that of the susceptible strain. In conclusion, increased abamectin resistance in B. praetiosa was associated with a rapid increase in MFOs activity, while GSTs also played a role in abamectin resistance development and RR strain exhibited reproductive disadvantages. [ABSTRACT FROM AUTHOR]

Tetranychus gloveri, a recently introduced mite species in Kerala, India has now established in the state as a predominant mite pest on ornamental plants. In this study, the status of acaricide resistance and the activity of detoxification enzymes were investigated in the populations of T. gloveri collected on Adenium plants from six different commercial horticultural nurseries of Thrissur district, Kerala, India. Adulticidal bioassays were performed for fenazaquin and dicofol, while nymphicidal bioassays were conducted for spiromesifen to estimate the LC50. The field populations showed resistance ratios in the range of 1.66–14.38-fold for fenazaquin, 1.06–27.31-fold for spiromesifen and 1.22–3.65-fold for dicofol, compared to the laboratory-maintained reference population. Enzyme assays of detoxifying enzymes revealed enhanced activity of carboxylesterase by 1.03–3.52-fold and cytochrome P450 monooxygenase by 1.01–2.08-fold in the field populations. However, the role of glutathione S-transferase in the development of resistance in different field populations was insignificant. The study recorded the development of resistance in the spider mite, T. gloveri to new generation acaricides spiromesifen and fenazaquin for the first time and also confirmed the significant role of the carboxylesterases and cytochrome P450 monooxygenases in imparting resistance in T. gloveri. [ABSTRACT FROM AUTHOR]

Background: The cabbage moth, Mamestra brassicae, is a polyphagous pest that attacks several crops. Here, the sublethal and lethal effects of chlorantraniliprole and indoxacarb were investigated on the developmental stages, detoxification enzymes, reproductive activity, calling behavior, peripheral physiology, and pheromone titer of M. brasssicae. Methods: To assess pesticide effects, the second instar larvae were maintained for 24 h on a semi-artificial diet containing insecticides at their LC10, LC30, and LC50 concentrations. Results: M. brassicae was more susceptible to chlorantraniliprole (LC50 = 0.35 mg/L) than indoxacarb (LC50 = 1.71 mg/L). A significantly increased developmental time was observed with both insecticides at all tested concentrations but decreases in pupation rate, pupal weight, and emergence were limited to the LC50 concentration. Reductions in both the total number of eggs laid per female and the egg viability were observed with both insecticides at their LC30 and LC50 concentrations. Both female calling activity and the sex pheromone (Z11-hexadecenyl acetate and hexadecenyl acetate) titer were significantly reduced by chlorantraniliprole in LC50 concentration. Antennal responses of female antennae to benzaldehyde and 3-octanone were significantly weaker than controls after exposure to the indoxocarb LC50 concentration. Significant reductions in the enzymatic activity of glutathione S-transferases, mixed-function oxidases, and carboxylesterases were observed in response to both insecticides. [ABSTRACT FROM AUTHOR]

In this study, responses of carboxylesterases, acetylcholinesterase, and stress protein Hsp70 were examined in the midgut and midgut tissue, and brain of fifth instar larvae of Lymantria dispar L. and Euproctis chrysorrhoea L. following chronic exposure to dietary fluoranthene. Specific carboxylesterase activity increased significantly in the midgut tissue of E. chrysorrhoea larvae treated with a lower fluoranthene concentration. The specific patterns of isoforms expression, recorded in larvae of both species, enable efficient carboxylesterase activity as a significant part of defense mechanisms. Increased Hsp70 concentration in the brain of L. dispar larvae points to a response to the proteotoxic effects of a lower fluoranthene concentration. Decreased Hsp70 in the brain of E. chrysorrhoea larvae in both treated groups can suggest induction of other mechanisms of defense. The results indicate the importance of the examined parameters in larvae of both species exposed to the pollutant, as well as their potential as biomarkers.

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. Hightemperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical a/ b-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal b-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. [ABSTRACT FROM AUTHOR]

The synthetic cannabinoid receptor agonists (SCRAs) (quinolin‐8‐yl 4‐methyl‐3‐(morpholine‐4‐sulfonyl)benzoate [QMMSB]) and (quinolin‐8‐yl 4‐methyl‐3‐((propan‐2‐yl)sulfamoyl)benzoate [QMiPSB], also known as SGT‐46) are based on the structure of quinolin‐8‐yl 4‐methyl‐3‐(piperidine‐1‐sulfonyl)benzoate (QMPSB) that has been identified on seized plant material in 2011. In clinical toxicology, knowledge of the metabolic fate is important for their identification in biosamples. Therefore, the aim of this study was the identification of in vitro Phase I and II metabolites of QMMSB and QMiPSB in pooled human liver S9 fraction (pHLS9) incubations for use as screening targets. In addition, the involvement of human monooxygenases and human carboxylesterases (hCES) was examined. Analyses were performed by liquid chromatography coupled with high‐resolution tandem mass spectrometry. Ester hydrolysis was found to be an important step in the Phase I metabolism of both SCRAs, with the carboxylic acid product being found only in negative ionization mode. Monohydroxy and N‐dealkyl metabolites of the ester hydrolysis products were detected as well as glucuronides. CYP2C8, CYP2C9, CYP3A4, and CYP3A5 were involved in hydroxylation. Whereas enzymatic ester hydrolysis of QMiPSB was mainly catalyzed by hCES1 isoforms, nonenzymatic ester hydrolysis was also observed. The results suggest that ester hydrolysis products of QMMSB and QMiPSB and their glucuronides are suitable targets for toxicological screenings. The additional use of the negative ionization mode is recommended to increase detectability of analytes. Different cytochrome P450 (CYP) isozymes were involved in the metabolism; thus, the probability of drug–drug interactions due to CYP inhibition can be assessed as low. [ABSTRACT FROM AUTHOR]

Horseradish peroxidase (HRP) combined with its fluorescence substrates is attracting increasing attention for biochemical analysis. Amplex red is the most widely used fluorescence substrate to HRP; however, it suffers from some drawbacks, such as nonspecific responsiveness toward carboxylesterases. Discovering a new small molecular fluorescence substrate with improved sensitivity and selectivity for HRP is thus desired. Herein, three dihydrofluorescein derivatives (DCFHs) are presented to serve as HRP substrates through fluorescence turn-on methods. The most promising one, 2,7-dichloro-9-(2-(hydroxymethyl)phenyl)-9H-xanthene-3,6-diol (DCFH-1), exhibited excellent sensitivity in the detection of HRP. Moreover, DCFH-1 does not respond to carboxylesterase, thus holding advantages over Amplex red. In the further study, the detection reagent in the commercial ELISA kits was replaced with DCFH-1 to establish a new fluorescence ELISA, which works very well in the quantification of inflammatory cytokine biomarkers from in vitro models. [ABSTRACT FROM AUTHOR]

Volatile ester compounds are important contributors to the flavor of strawberry, which affect consumer preference. Here, the GC-MS results showed that volatile esters are the basic aroma components of strawberry, banana, apple, pear, and peach, and the volatile esters were significantly accumulated with the maturation of strawberry fruits. The main purpose of this study is to discuss the relationship between carboxylesterases (CXEs) and the accumulation of volatile ester components in strawberries. FaCXE2 and FaCXE3 were found to have the activity of hydrolyzing hexyl acetate, Z-3-hexenyl acetate, and E-2-hexenyl acetate to the corresponding alcohols. The enzyme kinetics results showed that FaCXE3 had the higher affinity for hexyl acetate, E-2-hexenyl acetate, and Z-3-hexenyl acetate compared with FaCXE2. The volatile esters were mainly accumulated at the maturity stages in strawberry fruits, less at the early stages, and the least during the following maturation stages. The expression of FaCXE2 gradually increased with fruit ripening and the expression level of FaCXE3 showed a decreasing trend, which suggested the complexity of the true function of CXEs. The transient expression of FaCXE2 and FaCXE3 genes in strawberry fruits resulted in a significantly decreased content of volatile esters, such as Z-3-hexenyl acetate, methyl hexanoate, methyl butyrate, and other volatile esters. Taken together, FaCXE2 and FaCXE3 are indeed involved in the regulation of the synthesis and degradation of strawberry volatile esters. [ABSTRACT FROM AUTHOR]

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Pharmaceuticals such as antidepressants are designed to be bioactive at low concentrations. According to their mode of action, they can also influence non-target organisms due to the phylogenetic conservation of molecular targets. In addition to the pollution by environmental chemicals, the topic of microplastics (MP) in the aquatic environment came into the focus of scientific and public interest. The aim of the present study was to investigate the influence of the antidepressant amitriptyline in the presence and absence of irregularly shaped polystyrene MP as well as the effects of MP alone on juvenile brown trout (Salmo trutta f. fario). Fish were exposed to different concentrations of amitriptyline (nominal concentrations between 1 and 1000 µg/L) and two concentrations of MP (104 and 105 particles/L; <50 µm) for three weeks. Tissue cortisol concentration, oxidative stress, and the activity of two carboxylesterases and of acetylcholinesterase were assessed. Furthermore, the swimming behavior was analyzed in situations with different stress levels. Exposure to amitriptyline altered the behavior and increased the activity of acetylcholinesterase. Moreover, nominal amitriptyline concentrations above 300 µg/L caused severe acute adverse effects in fish. MP alone did not affect any of the investigated endpoints. Co-exposure caused largely similar effects such as the exposure to solely amitriptyline. However, the effect of amitriptyline on the swimming behavior during the experiment was alleviated by the higher MP concentration. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effects of europium (Eu) exposure (10 μg/L), warming (a 4 °C increase), and their combination on Mytilus galloprovincialis. Biochemical and histopathological changes in adult mussels were evaluated after a 28-day exposure period. Additionally, biochemical and physiological alterations in sperm were measured following a 30-min exposure period. The overall responses to each treatment were assessed using the Integrated Biological Response index version 2 (IBRv2). In adult mussels, warming elevated metabolism and activated glutathione S-transferases (GSTs), leading to redox imbalance and cellular damage. Europium exposure alone slightly enhanced metabolism and GSTs activity, resulting in cellular damage and histopathological injuries in digestive tubules. The combined exposure to Eu and warming was the most detrimental treatment for adults, as indicated by the highest IBRv2 value. This treatment slightly increased metabolism and uniquely elevated the activity of antioxidant enzymes, as well as GSTs and carboxylesterases. Despite these responses, they were inadequate to prevent redox imbalance, cellular damage, and histopathological injuries in digestive tubules and gills. Regarding sperm, warming reduced reactive oxygen species (ROS) production but raised lipid peroxidation levels. Sperm exposed to this treatment also increased their oxygen consumption and exhibited reduced velocity. The IBRv2 indicated that Eu was the most harmful treatment for sperm, significantly increasing ROS production and notably decreasing sperm velocity. When combined with warming, Eu elevated superoxide anion (O 2 -) production, lowered sperm velocity, and increased oxygen consumption. This study underscores the importance of investigating the effects of rare earth elements and their interaction with climate change-related factors. [Display omitted] • Antioxidant enzymes were only activated in adults exposed to the Eu and warming. • All treatments cause redox imbalance and cellular damage in adults. • Eu at both temperatures induced histopathological injuries in both tested organs. • All treatments caused a decrease in sperm velocity. • The worst treatment in adults was Eu and warming, while in sperm was Eu. [ABSTRACT FROM AUTHOR]

Quinolin-8-yl 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methylbenzoate (2F-QMPSB) and 3-(4,4-difluoropiperidine-1-sulfonyl)-4-methyl- N -(2-phenylpropan-2-yl)benzamide (SGT-233) belong to a new group of synthetic cannabinoid receptor agonists containing a sulfamoyl benzoate or sulfamoyl benzamide core structure. 2F-QMPSB was identified in herbal material seized in Europe in 2018. The aims of this study were the identification of in vitro Phase I and II metabolites of 2F-QMPSB and SGT-233 to find analytical targets for toxicological screenings. Furthermore, the contribution of different monooxygenases and human carboxylesterases to Phase I metabolism was investigated. Liquid chromatography coupled to high-resolution tandem mass spectrometry was used for analysis. Ester hydrolysis was found to be an important step in the metabolism of 2F-QMPSB, which was catalyzed mainly by human carboxylesterases (hCES)1 isoforms. Additionally, nonenzymatic ester hydrolysis was observed in case of 2F-QMPSB. Notably, the carboxylic acid product derived from ester hydrolysis and metabolites thereof were only detectable in negative ionization mode. In case of SGT-233, mono- and dihydroxy metabolites were identified, as well as glucuronides. The cytochrome P450 (CYP) isozymes CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 were found to be involved in the hydroxylation of both compounds. The results of these in vitro experiments suggest that the ester hydrolysis products of 2F-QMPSB and their glucuronides are suitable targets for toxicological screenings. In the case of SGT-233, the mono- and dihydroxy metabolites were identified as suitable screening targets. The involvement of various CYP isoforms in the metabolism of both substances reduces the likelihood of drug–drug interactions due to CYP inhibition. [ABSTRACT FROM AUTHOR]

Oedaleus asiaticus (Bey-Bienko) is an economically devastating locust species found in grassland and pastoral areas of the Inner Mongolia region of northern China. In this study, resistance to three frequently used insecticides (beta-cypermethrin, matrine, and azadirachtin) was investigated in six field populations of O. asiaticus using the leaf-dip bioassay method. The inhibitory effects of synergists and the activities of detoxification enzyme activities in the different populations were determined to explore potential biochemical resistance mechanisms. The results showed that the field populations SB (resistance ratio [RR] = 7.85), ZB (RR = 5.64), and DB (RR = 6.75) had developed low levels of resistance to beta-cypermethrin compared with a susceptible control strain. Both the SB (RR = 5.92) and XC (RR = 6.38) populations had also developed low levels of resistance against matrine, with the other populations remaining susceptible to both beta-cypermethrin and matrine. All field populations were susceptible to azadirachtin. Synergism analysis showed that triphenyl phosphate (TPP) and diethyl-maleate (DEM) increased the toxicity of beta-cypermethrin significantly in the SB population, while the synergistic effects of TPP, piperonyl butoxide (PBO), and DEM on the toxicity of matrine were higher in SB (SR 3.86, 4.18, and 3.07, respectively) than in SS (SR 2.24, 2.86, and 2.29, respectively), but no synergistic effects of TPP, PBO, and DEM on azadirachtin were found. Biochemical assays showed that the activities of carboxylesterases (CarEs) and glutathione- S -transferases (GSTs) were significantly raised in all field populations of O. asiaticus , with a significant positive correlation observed between beta-cypermethrin resistance and CarE activity. The activities of cytochrome P450 monooxygenases (P450) and multi-function oxidases (MFO) were elevated in all six field populations, and P450 activity displayed strong positive correlations with the three insecticides. Our findings suggest that resistance to beta-cypermethrin in O. asiaticus may be mainly attributed to elevated CarE and GST activities, while P450 plays an important role in metabolizing matrine and azadirachtin. Our study provides insights that will help improve insecticide resistance management strategies. [ABSTRACT FROM AUTHOR]

A prodrug approach is a powerful method to temporarily change the physicochemical and thus, pharmacokinetic properties of drugs. However, in site-selective targeted prodrug delivery, tissue or cell-specific bioconverting enzyme is needed to be utilized to release the active parent drug at a particular location. Unfortunately, ubiquitously expressed enzymes, such as phosphatases and carboxylesterases are well used in phosphate and ester prodrug applications, but less is known about enzymes selectively expressed, e.g., in the brain and enzymes that can hydrolyze more stable prodrug bonds, such as amides and carbamates. In the present study, L-type amino acid transporter 1 (LAT1)-utilizing amide prodrugs bioconverting enzyme was identified by gradually exploring the environment and possible determinants, such as pH and metal ions, that affect amide prodrug hydrolysis. Based on inducement by cobalt ions and slightly elevated pH (8.5) as well as localization in plasma, liver, and particularly in the brain, aminopeptidase B was proposed to be responsible for the bioconversion of the majority of the studied amino acid amide prodrugs. However, this enzyme hydrolyzed only those prodrugs that contained an aromatic promoiety (L-Phe), while leaving the aliphatic promoeities (L-Lys) and the smallest prodrug (with L-Phe promoiety) intact. Moreover, the parent drugs' structure (flexibility and the number of aromatic rings) largely affected the bioconversion rate. It was also noticed in this study, that there were species differences in the bioconversion rate by aminopeptidase B (rodents > human), although the in vitro-in vivo correlation of the studied prodrugs was relatively accurate. [ABSTRACT FROM AUTHOR]

Synthetic cannabinoids (SC) are new psychoactive substances known to cause intoxications and fatalities. One reason may be the limited data available concerning the toxicokinetics of SC, but toxicity mechanisms are insufficiently understood so far. Human carboxylesterases (hCES) are widely known to play a crucial role in the catalytic hydrolysis of drugs (of abuse). The aim of this study was to investigate the in vitro contribution of hCES to the metabolism of the 13 SC 3,5-AB-5F-FUPPYCA, AB-5F-P7AICA, A-CHMINACA, DMBA-CHMINACA, MBA-CHMINACA, MDMB-4F-BINACA, MDMB-4en-PINACA, MDMB-FUBICA, MDMB-5F-PICA, MMB-CHMICA, MMB-4en-PICA, MMB-FUBINACA, and MPhP-5F-PICA. The SC were incubated with recombinant hCES1b, hCES1c, or hCES2 and analyzed by liquid chromatography–ion trap mass spectrometry to assess amide or ester hydrolysis in an initial activity screening. Enzyme kinetic studies were performed if sufficient hydrolysis was observed. No hydrolysis of the amide linker was observed using those experimental conditions. Except for MDMB-5F-PICA, ester hydrolysis was always detected if an ester group was present in the head group. In general, SC with a terminal ester bearing a small alcohol part and a larger acyl part showed higher affinity to hCES1 isozymes. Due to the low hydrolysis rates, enzyme kinetics could not be modeled for the SC with a tert-leucine-derived moiety, but hydrolysis reactions of MPhP-5F-PICA and of those containing a valine-derived moiety followed classic Michaelis–Menten kinetics. In conclusion, drug–drug/drug–food interactions or hCES polymorphisms may prolong the half-life of SC and the current results help to estimate the risk of toxicity in the future after combining them with activity and clinical data. [ABSTRACT FROM AUTHOR]

Esterases represent a class of hydrolases, and are widely distributed in animals, plants and microorganisms. Depending on whether they hydrolyze small water-soluble esters or ester bonds of long-chain acylglycerols, they are classified as carboxylesterases (EC 3.1.1.1) or lipases (EC 3.1.1.3). A host of novel microbial carboxylesterases from culture-independent methods have been also discovered and overexpressed within the last few years. Indeed, genome mining has emerged as an inexpensive and readily available bioinformatics technique to unveil novel enzymes and natural products, based on the homologies of pre-identified genomic information, without the limitations associated with traditional culturing techniques. This review aims to give an overview of the recently discovered carboxylesterases via genome mining, their structural features, and reaction mechanism with specific emphasis on their roles in chemical kinetic resolution of compounds with pharmaceutical and biotechnological applications. • Newly discovered carboxylesterases via metagenomics are reviewed. • The biochemical information as well as characterization of discovered carboxylesterases are discussed. • Carboxylesterase role in chemical kinetic resolution of the bioactive valuable products is explained. • The stereo-preference of carboxylesterases has been investigated with the aid of in silico studies. [ABSTRACT FROM AUTHOR]

Microplastics (MPs) are critical emerging pollutants around the world. There is a growing interest in the effects of MP ingestion, non-digestion, and toxicity on aquatic organisms. Amphibian tadpoles are the vertebrate group that has received the least attention regarding this issue. The aim of the present study was to determine the ingestion of polyethylene MPs by Scinax squalirostris tadpoles by atomic force microscopy (AFM) and to evaluate the activities of carboxylesterase (CbE, using 4-naphthyl butyrate-NB-, and 1-naphthyl acetate –NA- as substrates) and alkaline phosphatase (ALP) under MP exposure. Enzyme activities were analyzed spectrophotometrically at 2 and 10 days of exposure. Tadpoles were exposed to two different treatments during 10 days: a negative control (CO, dechlorinated water) and MP (60 mg L−1). AFM images of the digestive contents of tadpoles revealed the presence of MPs. After 10 days of MP exposure, CbE (NB) activity was significantly higher and CbE (NA) activity was significantly lower in MP treatments than in controls. ALP activity decreased in MP treatments after 2 and 10 days of exposure. The detection of MP particles in the intestinal contents and the effects on metabolic enzymes in a common frog species evidenced the potential health risk of MP to aquatic vertebrates. Thus, the differential response in enzymes and substrates demonstrate the need for considering the complex effects of contaminants and nutrients on ecosystems for ecotoxicological risk characterization. [ABSTRACT FROM AUTHOR]

Plutella xylostella (L.) (Lepidoptera: Plutellidae) is one of the most destructive pests of Brassicaceae vegetables. Cantharidin is an insect-derived defensive toxin, which has been reported to have toxicity to a variety of pests and especially lepidopteran pests. Although the toxicity of cantharidin on P. xylostella has been demonstrated, there is little information available on the specific detoxification response of P. xylostella against cantharidin. This study investigates the enzymatic response (including serine/threonine phosphatases [PSPs], carboxylesterases [CarEs], glutathione-S-transferases [GSTs], and cytochrome P450 monooxygenases [P450]) in P. xylostella to the sublethal and low lethal concentrations of cantharidin (LC10 and LC25). Results showed that the inhibitory activity of PSPs was increased and then decreased in vivo, while PSPs activity could be almost completely inhibited in vitro. Interestingly, the activities of detoxification enzymes (GST, CarE, and P450) in P. xylostella displayed a trend of decreasing and then increasing after exposure to the two concentrations of cantharidin. Notably, the increase in P450 enzyme activity was the most significant. The increasing trend of detoxification enzyme activity was congruent with the recovery trend of PSPs activity. This study contributes to our understanding of the detoxification mechanism of cantharidin in P. xylostella and helps in the further development of biogenic agents. [ABSTRACT FROM AUTHOR]