Wolbachia-mediated regulation of EscrGST1 modulates pesticide resistance in Eucryptorrhynchus scrobiculatus.

Self-assembled microalgae-photosensitized biohybrids enabling solar-driven directed phosphate conversion form malathion.

Sexual signalling by pheromones is essential for mate finding and mate choice in moths and plays an important role in reproductive isolation. Acetates (i.e., acetate esters) produced by females of Heliothis (Chloridea) subflexa Fabricius, 1777 (Lepidoptera: Noctuidae) attract conspecific males but repel Heliothis virescens Fabricius, 1777 (Lepidoptera: Noctuidae) males. A QTL (quantitative trait locus) harbouring carboxylesterases and lipases was previously shown to affect acetates, and CRISPR/Cas9-induced knockouts increased acetate amounts by blocking hydrolysis of the esters as expected. A second, unlinked QTL, containing a cluster of three different carboxylesterases (CXEs), unexpectedly yielded decreased acetate amounts. In one of these genes, esterase CXE24, we found a naturally occurring transposable element insertion in exon 8. A CRISPR/Cas9-induced frameshift at the same position yielded the same results. The paradox was resolved by a CRISPR/Cas9-induced frameshift in exon 2 of CXE24 which increased acetate amounts. The frameshift in exon 2 produced a truncated protein lacking the substrate binding site and the catalytic triad, while the frameshift in exon 8 removed only the third residue of the catalytic triad. In silico modelling showed that the exon-8-truncated protein could not hydrolyse the esters by itself, which likely explains the decreased acetate amounts. To place our findings in an evolutionary context, we explored variation in the esterase cluster in 16 species of Lepidoptera with completely sequenced genomes. Geographic and temporal variation in acetates has been observed in H. subflexa, and variation in the frequency of the transposable element could be a possible explanation.

Trace metals and metalloids pose persistent threats to freshwater ecosystems, yet their trophic transfer and sublethal effects across food webs remain poorly understood. We investigated bioaccumulation patterns and biomarker responses in a predator–prey system comprising Prussian carp (Carassius gibelio) and Great Cormorant (Phalacrocorax carbo) nestlings from the Danube floodplain wetland Kopački rit Nature Park (Croatia) during 2023–2024. Concentrations of arsenic (As), selenium (Se), cadmium (Cd), mercury (Hg) and lead (Pb) were determined in Prussian carp liver and in Great Cormorant whole blood. The activities of acetylcholinesterase (AChE), carboxylesterase (CES), glutathione S-transferase (GST) and the levels of reactive oxygen species (ROS) and reduced glutathione (GSH) were measured in brain, muscle and gill tissues of Prussian carp, as well as in plasma and S9 blood fractions of Great Cormorants. In addition, tissue-specific metal concentration ratios (TSMCR) were calculated to assess the relative magnitude of recent dietary exposure in the predator compared to the prey. Biomarker activity showed strong tissue- and fraction-specific variation, with temporal differences. Exposure–response modelling revealed significant associations between As, Cd, and Hg and specific biomarkers, particularly in gill and plasma. Cross-species comparisons indicated elevated TSMCR as a proxy for recent trophic exposure only for Hg in 2023, whereas As and Se exhibited lower TSMCR. These findings demonstrate that metal exposure in floodplain systems induces physiological responses and Hg poses the greatest prey-to-predator exposure risk, highlighting the value of integrating pollutant measurements with mechanistic biomarker endpoints to evaluate ecosystem-level impacts.

Background/Objectives: This study focused on synthesizing novel alkenyl derivatives of azinylferrocenes and evaluating their potential as Alzheimer’s disease (AD) therapeutics. Methods: 1-Azinyl-1′-acetylferrocenes were obtained by regioselective acetylation of azinylferrocenes, followed by the Wittig reaction or reduction of 1-azinyl-1′-acetylferrocenes and subsequent dehydration of the resulting alcohols. The synthesized compounds underwent the following biological activity testing relevant to AD: inhibition of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and off-target carboxylesterase (CES); antioxidant capacity (ABTS and FRAP assays); inhibition of Aβ42 self-aggregation (thioflavin method); blocking AChE-induced β-amyloid aggregation (propidium displacement); and cytotoxicity in SH-SY5Y and MSC-Neu cells (MTT assay). Results: Quinoline and bipyridine derivatives demonstrated effective cholinesterase inhibition, especially quinoline 7b (AChE IC50 3.32 μM; BChE IC50 3.68 μM), while acridine derivatives were poor inhibitors. Quantum chemical (QC) calculations predicted that acridine derivatives were especially prone to form stable dimers. Molecular docking into protein targets generated by an AlphaFold3 reproduction code showed that these dimers were too bulky to access enzyme active sites, yet they could bind to protein surfaces to inhibit Aβ42 self-aggregation and displace propidium from the AChE peripheral anionic site. All compounds showed high antioxidant activity in ABTS and FRAP assays, with quinoline derivatives being 2–4 times more potent than Trolox. QC calculations supported these findings. Quinoline and bipyridine derivatives also exhibited low cytotoxicity and scant CES inhibition. Conclusions: Overall, the synthesized ferrocenes, particularly the quinoline and bipyridine derivatives, appear promising for further research as multifunctional therapeutic agents targeting AD due to their anticholinesterase, antiaggregating, and antioxidant activities combined with low toxicity.

The abnormally high expression of glutathione (GSH) in hepatocellular carcinoma (HCC) cells significantly contributes to chemotherapy resistance by neutralizing reactive oxygen species (ROS). To address this problem, we developed a nanocomposite probe (micelle@PDA@MnO2 NPs) which was composed of free radical-labeled micelles as the core, polydopamine (PDA) as the inner shell and MnO2 nanosheets as the outer shell. In the HCC microenvironment, the over-expressed GSH triggered the decomposition of the MnO2 shell, releasing Mn2+ ions, and subsequently, the acidic condition and over-expressed carboxylesterase 2 (CES2) worked together to mediate the controlled release of nitroxide free radicals (i.e., TEMPONE) from the micelle core. With the help of dual-signal electron spin resonance (ESR) detection, it was found that much more free radicals and Mn2+ were released in hepatoma cells than in normal liver cells, rendering the proposed nanotherapeutic platform specific to HCC and a good candidate for free radical therapy and chemodynamic therapy (CDT). Moreover, the therapy featured an intelligent self-amplified property: the abundant GSH in the tumor microenvironment rapidly activated the decomposition of the nanocomposite and triggered the release of free radical TEMPONE and Mn2+, and TEMPONE and Mn2+ further amplified the oxidative stress to induce tumor apoptosis via ROS generation and GSH depletion. Finally, in combination with three therapy modes (Mn2+-mediated CDT, the oxidative stress enhancement effect of TEMPONE, and PDA-based photothermal therapy), significant therapeutic effects including facilitation of HepG2 cell apoptosis and inhibition of tumor growth in the HCC mouse model were achieved.

Fluorescein diacetate (FDA) should not be used to study human carboxylesterase 2 (CES2) in complex biological systems without validation.

Diaphorina citri is one of the most severe pests affecting citrus production worldwide. Our previous research identified a series of quassinoids that exhibit insecticidal activity against D. citri adults. However, the larvae of D. citri also pose a significant threat to the citrus industry. In an effort to discover novel larvicidal lead compounds, a systematic phytochemical study on the relatively understudied fruits of Picrasma quassioides was conducted, and the insecticidal mechanism was also explored preliminarily. Ten new (1-10) and 15 known quassinoids (11-25) were isolated from P. quassioides fruits. All these new compounds possess a hydroxyl group at C-14 except for compound 5, which is a rare example of quassinoid with a double bond located at C-14 and C-15. In terms of biological actions, compounds 2, 3, 5, 7, 10-16 and 18-23 had potent insecticidal activity. Preliminary structure and activity relationship (SAR)studies have shown that the insecticidal activity of quassinoids is closely related to the types of substituents at positions C-12, C-14 and C-16. Real-time quantitative polymerase chain reaction analysis demonstrated that compounds 7, 11 and 12 disturbed the expression of cytochrome P450, carboxylate esterase and glutathione S-transferase genes. Quassinoids isolated from fruits of P. quassioides demonstrate significant efficacy in managing the larvae of D. citri. These active compounds may serve as crucial lead structures for the development of highly effective plant-derived insecticides and control citrus Huanglongbing which mainly transmitted by D. citri. © 2025 Society of Chemical Industry.

Seasonal influence on the microbial diversity and flavor substances in the strong flavor Daqu fermentation

ABSTRACT Alkylphenols (APs) and their ethoxylates (APEs) are endocrine disruptors found in aquatic environments. This study evaluated the neurotoxic effects of bisphenol A (BPA), 4-nonylphenol (4-NP), and 3-tert-butylphenol (tertBP) in wild Mezquital silverside (Chirostoma jordani) from Lake Zumpango across dry, rainy, and cold-dry seasons. Biomarkers analyzed included acetylcholinesterase (AChE), carboxylesterase (CbE), butyrylcholinesterase (BChE), and γ-aminobutyric acid (GABA) levels in brain and spinal cord. Results showed seasonal and tissue-specific differences in enzymatic activities correlating with AP and APE concentrations. BChE and CbE activities were elevated during the dry season, associated with higher 4-NP sediment levels and lower BPA and tertBP in water. AChE activity and GABA levels peaked in the rainy season when tertBP was absent. Total phenols fluctuated seasonally but showed no clear neurotoxic association. These findings provide initial evidence of neurotoxic disruption in wild Mezquital silverside by BPA, 4-NP, and tertBP, warranting further ecotoxicological investigation.

Olfactory perception of trifluralin by GOBP2 decreases the susceptibility of Spodoptera litura to insecticides through modulation of 20E signaling pathway-mediated metabolic detoxification.

Background/Objectives: Metabolic enzymes are crucial for the detoxification of exogenously administered drugs, especially enzymes expressed in the intestine and the liver. Recent advancements in analytical methodologies enable sensitive and specific quantitative measurements of proteins, facilitating a more accurate evaluation of their expression and relative contribution to drug metabolism. Methods: The aim of the study was to characterize the protein expression levels of 16 Cytochrome P450s (CYP450s) and 2 carboxylesterases (CESs) in human liver and intestinal tissues using absolute quantification by HPLC-MS/MS. Human hepatocytes (HHEP) and human liver microsomes (HLM) were utilized, along with a novel intestinal preparation from cryopreserved human intestinal mucosa (CHIM), to perform proteomic analyses. Results: A comprehensive evaluation of 16 CYP450s and 2 CES enzyme expression in human liver and intestinal tissues is provided to reflect their relative abundance. Among the various in vitro systems evaluated, 14 of 16, 15/16, and 7/16 CYP450 of the isoforms analyzed were detected in HHEP, HLM, and CHIM, respectively. In hepatic systems, CYP2C9 exhibited the highest expression among CYP450 isoforms, a trend consistently observed in both HHEP and HLM. CYP3A4 was the most abundantly expressed isoform in CHIM preparations. Across all systems tested, CES1 and CES2 showed the highest overall protein expression levels, surpassing those of the CYP450s. Conclusions: Our findings demonstrate that the absolute quantification method employed is reliable, producing consistent results across two different in vitro hepatic systems (HHEP and HLM). This study supports the utility of absolute quantification approaches for accurately profiling drug-metabolizing enzymes and provides new, valuable insights to improve in vitro/in vivo extrapolation and more informed predictive pharmacokinetic modeling strategies.

SummaryIrinotecan (CPT-11) is a key chemotherapeutic agent for gastric cancer (GC), but its efficacy is limited by variable patient responses. The activation of CPT-11 to its active form, SN-38, depends on the enzyme carboxylesterase 2 (CES2), yet the regulation of CES2 in GC is poorly understood. Here, we identify Krüppel-like factor 13 (KLF13) as a direct transcriptional activator of CES2. We show that KLF13 expression correlates with CES2 levels and treatment response in GC patients. Mechanistically, KLF13 binds to specific elements in the CES2 promoter to drive its expression. This function of KLF13 is critically dependent on its acetylation by the co-activator p300, as demonstrated by site-directed mutagenesis and p300 inhibition. Functionally, the KLF13-CES2 axis dictates cellular sensitivity to CPT-11. Our findings unveil a p300-KLF13-CES2 signaling pathway that governs irinotecan sensitivity, providing potential predictive biomarkers and therapeutic targets for GC.

Clopidogrel, a frequently used prodrug, is convertedto its activemetabolite through the intermediate 2-oxo-clopidogrel by cytochromeP450 (CYP) enzymes, which accounts for only 5%–15% of its metabolism.Majority of the clopidogrel dose (85%–90%) is extensively hydrolyzedto its inactive metabolite, clopidogrel carboxylic acid by carboxylesterase1 (CES1). In vitro studies suggest the involvement of multiple CYPisoforms, with CYP1A2, CYP2C19, and CYP2B6 identified as major contributorsto 2-oxo-clopidogrel formation. While CYP2C19 genetic polymorphismsare often highlighted as the primary factor contributing to variabilityin the clopidogrel response, the confounding role of CES1 variabilityon clopidogrel oxidation is less well understood. Our study utilizingproteomics-informed scaling highlights the importance of accurateestimation of the fraction metabolized (fm) by CES1 and CYPs in clopidogrel metabolism. The results also indicatethat differential subcellular localization of these enzymes and technicalvariability in sample preparation can influence fm estimation, suggesting that HLM may not be an idealmodel for investigating dual substrates of CYPs and CES. Quantitativeproteomics and activity assays revealed significant variability inthe absolute content and activities of CES1 and CYP enzymes acrossHLM donors (n = 10), which affected the estimationof fmCES versus fmCYP. Human hepatocyte assay, which represents a CYP versusCES abundance ratio similar to that in liver tissue, demonstratedthe critical roles of CYP3A4 and CES1 abundance in the 2-oxo-clopidogrelformation rate. Further, enzyme kinetic studies identified CYP3A4as the primary contributor to 2-oxo-clopidogrel formation, but multipleother enzymes, including CYP2C9, were identified as contributors.Overall, our findings emphasize the need for accounting for variabilityin both CES1 and CYP enzymes to improve fm estimation in the in vitro to in vivo extrapolation of dual substratesof CYP/CES such as clopidogrel.

Polyphagous insects utilize carboxylesterases (COEs) to defense against plant allelochemicals, though the mechanisms underlying COE-mediated detoxification remain elusive. Using the xanthotoxin-Spodoptera litura model, we reveal a detoxification framework integrating hormonal regulation and catalytic plasticity. The 20-hydroxyecdysone receptor EcR/USP coordinates stage-specific COE induction during feeding phases, as evidenced by 30.87-43.21% COE052 suppression post-RNAi and increased larval xanthotoxin susceptibility. Promoter activation assays identified EcR/USP-responsive elements, with mutagenesis revealing the critical -495/-481 regulatory sequence in COE052. Functionally, COE052 demonstrates dual functionality, achieving 31.41% xanthotoxin degradation concurrent with 44.34% ROS reduction. Structural analyses characterized three noncatalytic residues (Gly130/Thr463/His464) as an allosteric gatekeeper, whose mutations reduced metabolic efficiency (16.83-25.83%) via substrate-network destabilization. Furthermore, all mutants displayed impaired oxidative stress tolerance in prokaryotic systems, validating synergistic roles in redox maintenance. These discoveries culminate in a tripartite defense model integrating hormonal coordination, transcriptional adaptability, and structural evolution, wherein noncatalytic domains critically potentiate COE052's functional spectrum.

Harnessing the potential of a carboxylesterase-responsive chemiluminescent probe for the diagnosis and therapeutic assessment of ALI.

Pharmacokinetics and pre-systemic biotransformation of a deuterated Clopidogrel-Ferulic acid conjugate in rats.

Castration-resistant prostate cancer (CRPC) remains difficult to treat with conventional chemotherapy. We evaluated a stem cell-based enzyme-prodrug strategy using hTERT-immortalized adipose-derived stem cells engineered to express rabbit carboxylesterase 2 (hTERT-ADSC.CE2) in combination with irinotecan (CPT-11). hTERT-ADSC.CE2 cells were generated via lentiviral transduction and confirmed to overexpress CE2. Their tumor-homing capacity toward PC3 prostate cancer cells was assessed, along with prodrug activation, apoptosis induction, and in vivo tumor suppression in a CRPC mouse model. hTERT-ADSC.CE2 cells demonstrated enhanced migration toward PC3 cells and higher expression of tumor-homing factors than the controls. Under CPT-11, they exhibited a strong “suicide” effect and induced selective killing of PC3 cells, with upregulation of BAX and cleaved caspase-3 and downregulation of BCL-2. By day 14, the combination arm showed significantly lower tumor burden than both the control and irinotecan-alone arms (p < 0.05). The pattern is consistent with intratumoral activation and localized SN-38 exposure. hTERT-ADSC.CE2 combined with irinotecan provides potent, tumor-targeted cytotoxicity and markedly suppresses CRPC progression. This cell-mediated prodrug activation system may represent a promising therapeutic approach for advanced prostate cancer.

Malathion has the reputation of being a safe pesticide. There are no reported cases of cholinergic toxicity in people exposed to low environmental doses of malathion. Our goal was to explain the safety of malathion in terms of the mechanism of malathion detoxication. The structure of malathion includes a built-in safety feature, specifically two ethyl esters. The ethyl esters are decarboxylated by human esterases to negatively charged malathion which does not react with acetylcholinesterase. Acetylcholinesterase is the toxicologically relevant target for organophosphates such as malathion. A toxic form of malathion is produced by Cytochrome P450 enzymes which convert malathion to malaoxon. Malaoxon is toxic because it inhibits acetylcholinesterase. We used high pressure liquid chromatography on a Prodigy 5 µm ODS column to monitor the production of enzyme-catalyzed decarboxylation of the malathion ethyl esters. The products of malathion decarboxylation were identified by mass spectrometry using a Thermo RSLC Ultimate 3000 ultra-high pressure chromatography system with a Thermo Easy-Spray PepMap RSLC C18 separation column attached to an Orbitrap Fusion Lumos Tribrid mass spectrometer. Decarboxylation and enzyme inhibition were assayed with recombinant human acetylcholinesterase (rHuAChE), human butyrylcholinesterase (HuBChE), and recombinant human liver carboxylesterase (rHuCE1). A trace contaminant in 98.5% pure malathion was identified by mass spectrometry. Consistent with the fact that negatively charged compounds are not inhibitors of HuAChE, HuBChE, or HuCE1, we found that negatively charged, decarboxylated malathion did not inhibit the activity of rHuAChE, HuBChE, or rHuCE1. Carboxylesterase detoxified malathion 100,000-fold faster compared to rHuAChE and HuBChE. Low dose exposures to malathion are not directly toxic The toxic metabolite, malaoxon, is produced very slowly. By comparison, detoxified malathion acids are formed rapidly. In conclusion, our data suggest that the safety of low dose environmental exposures to malathion is explained by the fact that malathion is detoxified faster than it is activated to the toxic malaoxon. Our review of the literature finds no convincing evidence that low dose malathion exposure causes cancer.

Photodynamic therapy (PDT) faces significant challenges in treating deep tumors due to the limited light penetration depth. Cerenkov radiation-induced PDT (CR-PDT) offers a potential solution by harnessing luminescence derived from radionuclides. However, the simultaneous delivery of radionuclides and photosensitizers in conventional CR-PDT systems leads to unnecessary phototoxicity of healthy tissues. Here, we present a tumor microenvironment (TME)-activated, dual-locked nanophotosensitizer (89Zr-PHZ-BrCyE) that integrates 89Zr-labeled pH-responsive polymeric micelle and a carboxylesterase (CrES)-activated photosensitizer prodrug for precise deep tumor therapy. The dual-responsive CR-PDT system demonstrated highly selective cytotoxicity toward hepatocellular carcinoma (HepG2) cells, with minimal impact on normal liver cells (L02). Upon intravenous injection, 89Zr-PHZ-BrCyE exhibited robust tumor inhibition and excellent biosafety in both subcutaneous and orthotopic H22 tumor models. This approach holds great promise for improving the therapeutic outcomes of CR-PDT in deep-seated tumors while ensuring good biosafety, paving the way for future potential clinical applications in the future.