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The efficiency of the Fenton reaction is markedly contingent upon the operational pH related to iron solubility. Therefore, a heterogeneous Fenton reaction has been developed to function at neutral pH. In the present study, the Bio-Fenton reaction was carried out using magnetite (Fe(II)Fe(III) 2 O 4 ) and H 2 O 2 generated by a newly isolated H 2 O 2 -producing bacterium, Desemzia sp. strain C1 at pH 6.8 to degrade chloroacetanilide herbicides. The optimal conditions for an efficient Bio-Fenton reaction were 10 mM of lactate, 0.5% (w/v) of magnetite, and resting-cells (O.D. 600  = 1) of strain C1. During the Bio-Fenton reaction, 1.8-2.0 mM of H 2 O 2 was generated by strain C1 and promptly consumed by the Fenton reaction with magnetite, maintaining stable pH conditions. Approximately, 40-50% of the herbicides underwent oxidation through non-specific reactions of •OH, leading to dealkylation, dechlorination, and hydroxylation via hydrogen atom abstraction. These findings will contribute to advancing the Bio-Fenton system for non-specific oxidative degradation of diverse organic pollutants under in-situ environmental conditions with bacteria producing high amount of H 2 O 2 and magnetite under a neutral pH condition., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The lack of data on the chronic effects of chloroacetanilide herbicide metabolites on non-target aquatic organisms creates a gap in knowledge about the comprehensive impacts of excessive and repeated pesticide use. Therefore, this study evaluates the long-term effects of propachlor ethanolic sulfonic acid (PROP-ESA) after 10 (T1) and 20 (T2) days at the environmental level of 3.5 μg.L -1 (E1) and its 10x fold multiply 35 μg.L -1 (E2) on a model organism Mytilus galloprovincialis. To this end, the effects of PROP-ESA usually showed a time- and dose-dependent trend, especially in its amount in soft mussel tissue. The bioconcentration factor increased from T1 to T2 in both exposure groups - from 2.12 to 5.30 in E1 and 2.32 to 5.48 in E2. Biochemical haemolymph profile and haemocyte viability were not affected by PROP-ESA exposure. In addition, the viability of digestive gland (DG) cells decreased only in E2 compared to control and E1 after T1. Moreover, malondialdehyde levels increased in E2 after T1 in gills, and DG, superoxidase dismutase activity and oxidatively modified proteins were not affected by PROP-ESA. Histopathological observation showed several damages to gills (e.g., increased vacuolation, over-production of mucus, loss of cilia) and DG (e.g., growing haemocyte trend infiltrations, alterations of tubules). This study revealed a potential risk of chloroacetanilide herbicide, propachlor, via its primary metabolite in the Bivalve bioindicator species M. galloprovincialis. Furthermore, considering the possibility of the biomagnification effect, the most prominent threat poses the ability of PROP-ESA to be accumulated in edible mussel tissues. Therefore, future research about the toxicity of pesticide metabolites alone or their mixtures is needed to gain comprehensive results about their impacts on living non-target organisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Memory deficits are a debilitating symptom of epilepsy, but little is known about mechanisms underlying cognitive deficits. Here, we describe a Na+ channel-dependent mechanism underlying altered hippocampal dendritic integration, degraded place coding and deficits in spatial memory. Two-photon glutamate uncaging experiments revealed a marked increase in the fraction of hippocampal first-order CA1 pyramidal cell dendrites capable of generating dendritic spikes in the kainate model of chronic epilepsy. Moreover, in epileptic mice dendritic spikes were generated with lower input synchrony, and with a lower threshold. The Nav1.3/1.1 selective Na+ channel blocker ICA-121431 reversed dendritic hyperexcitability in epileptic mice, while the Nav1.2/1.6 preferring anticonvulsant S-Lic did not. We used in vivo two-photon imaging to determine if aberrant dendritic excitability is associated with altered place-related firing of CA1 neurons. We show that ICA-121431 improves degraded hippocampal spatial representations in epileptic mice. Finally, behavioural experiments show that reversing aberrant dendritic excitability with ICA-121431 reverses hippocampal memory deficits. Thus, a dendritic channelopathy may underlie cognitive deficits in epilepsy and targeting it pharmacologically may constitute a new avenue to enhance cognition., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain.)

The metabolism and toxicity of current-use herbicide safeners remain understudied. We investigated the enantioselective metabolism of the safener benoxacor in Rhesus monkey subcellular fractions. Benoxacor was incubated with liver microsomes and cytosol from female and male monkeys (≤30 min). Benoxacor levels and enantiomeric fractions were determined with gas chromatography. Benoxacor was metabolized by microsomal cytochrome P450 enzymes (CYPs), cytosolic glutathione-S-transferases (GSTs), and microsomal and cytosolic carboxylesterase (CESs). CES-mediated microsomal metabolism followed the order males > females, whereas the CYP-mediated clearance followed the order females > males. CYP-mediated metabolism initially resulted in an enrichment of the second eluting benoxacor enantiomer (E 2 -benoxacor), whereas the first eluting benoxacor enantiomer (E 1 -benoxacor) was enriched after 10 or 30 min in female or male microsomal incubations. Benoxacor metabolism by GSTs was enantiospecific, with a total depletion of E 1 -benoxacor after approximately 20 min. Thus, the enantioselective metabolism of benoxacor by GSTs and CYPs may affect its toxicity., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hans-Joachim Lehmler reports financial support was provided by National Institute of Environmental Health Sciences. David Cwiertny reports financial support was provided by National Science Foundation. Hans-Joachim Lehmler reports financial support was provided by National Science Foundation., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Glioma-associated microglia and macrophages (GAMMs) are key players in creating an immunosuppressive microenvironment. They can be efficiently targeted by inhibiting the colony-stimulating factor 1 receptor (CSF-1R). We applied noninvasive PET/CT and PET/MRI using 18 F-fluoroethyltyrosine ( 18 F-FET) (amino acid metabolism) and N,N -diethyl-2-[4-(2- 18 F-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5- a ]pyrimidine-3-acetamide ( 18 F-DPA-714) (translocator protein) to understand the role of GAMMs in glioma initiation, monitor in vivo therapy-induced GAMM depletion, and observe GAMM repopulation after drug withdrawal. Methods: C57BL/6 mice ( n = 44) orthotopically implanted with syngeneic mouse GL261 glioma cells were treated with different regimens using the CSF-1R inhibitor PLX5622 (6-fluoro- N -((5-fluoro-2-methoxypyridin-3-yl)methyl)-5-((5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)methyl)pyridin-2-amine) or vehicle, establishing a preconditioning model and a repopulation model, respectively. The mice underwent longitudinal PET/CT and PET/MRI. Results: The preconditioning model indicated similar tumor growth based on MRI (44.5% ± 24.8%), 18 F-FET PET (18.3% ± 11.3%), and 18 F-DPA-714 PET (16% ± 19.04%) volume dynamics in all groups, suggesting that GAMMs are not involved in glioma initiation. The repopulation model showed significantly reduced 18 F-DPA-714 uptake (-45.6% ± 18.4%), significantly reduced GAMM infiltration even after repopulation, and a significantly decreased tumor volume (-54.29% ± 8.6%) with repopulation as measured by MRI, supported by a significant reduction in 18 F-FET uptake (-50.2% ± 5.3%). Conclusion: 18 F-FET and 18 F-DPA-714 PET/MRI allow noninvasive assessment of glioma growth under various regimens of CSF-1R therapy. CSF-1R-mediated modulation of GAMMs may be of high interest as therapy or cotherapy against glioma., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Studies on colony-stimulating factor 1 receptor (CSF-1R) inhibition-induced microglia depletion indicated that inhibitor withdrawal allowed the renewal of the microglia compartment via repopulation and resolved the inflammatory imbalance. Therefore, we investigated for the first time (to our knowledge) the effects of microglia repopulation on inflammation and functional outcomes in an ischemic mouse model using translocator protein (TSPO)-PET/CT and MR imaging, ex vivo characterization, and behavioral tests. Methods: Eight C57BL/6 mice per group underwent a 30-min transient occlusion of the middle cerebral artery. The treatment group received CSF-1R inhibitor in 1,200 ppm PLX5622 chow (Plexxikon Inc.) from days 3 to 7 to induce microglia/macrophage depletion and then went back to a control diet to allow repopulation. The mice underwent T2-weighted MRI on day 1 after ischemia and 18 F-labeled N,N -diethyl-2-(2-[4-(2-fluoroethoxy)phenyl]-5,7-dimethylpyrazolo[1,5-α]pyrimidine-3-yl)acetamide ( 18 F-DPA-714) (TSPO) PET/CT on days 7, 14, 21, and 30. The percentage injected tracer dose per milliliter within the infarct, contralateral striatum, and spleen was assessed. Behavioral tests were performed to assess motor function recovery. Brains were harvested on days 14 and 35 after ischemia for ex vivo analyses (immunoreactivity and real-time quantitative polymerase chain reaction) of microglia- and macrophage-related markers. Results: Repopulation significantly increased 18 F-DPA-714 uptake within the infarct on days 14 ( P  < 0.001) and 21 ( P  = 0.002) after ischemia. On day 14, the ionized calcium binding adaptor molecule 1 (Iba-1)-positive cell population showed significantly higher expression of TSPO, CSF-1R, and CD68, in line with microglia repopulation. Gene expression analyses on day 14 indicated a significant increase in microglia-related markers ( csf-1r, aif1, and p2ry12 ) with repopulation, whereas peripheral cell recruitment-related gene expression decreased ( cx3cr1 and ccr2 ), indicative of peripheral recruitment during CSF-1R inhibition. Similarly, uncorrected spleen uptake was significantly higher on day 7 after ischemia with treatment ( P  = 0.001) and decreased after drug withdrawal. PLX5622-treated mice walked a longer distance ( P  < 0.001) and more quickly ( P  = 0.009), and showed greater forelimb strength ( P  < 0.001), than control mice on day 14. Conclusion: This study highlighted the potential of 18 F-DPA-714 PET/CT imaging to track microglia and macrophage repopulation after short-term CSF-1R inhibition in stroke., (© 2022 by the Society of Nuclear Medicine and Molecular Imaging.)

Chloroacetamide herbicides (CAAHs) are important herbicides that were widely used to control agricultural weeds. However, their mass applications have seriously contaminated environment, and they are toxic to living beings. CAAHs are easy to enter anoxic environments such as subsoil, wetland sediment, and groundwater, where CAAHs are mainly degraded by anaerobic organisms. To date, there are no research on the anaerobic degradation of CAAHs by pure isolate and toxicity of anaerobic metabolites of CAAHs. In this study, the anaerobic degradation kinetics and metabolites of CAAHs by an anaerobic isolate BAD-10 T and the toxicity of anaerobic metabolites were studied. Isolate BAD-10 T could degrade alachlor, acetochlor, propisochlor, butachlor, pretilachlor and metolachlor with the degradation kinetics fitting the pseudo-first-order kinetics equation. The degradation rates of CAAHs were significantly affected by the length of N-alkoxyalkyl groups, the shorter the N-alkoxyalkyl groups, the higher the degradation rates. Four metabolites 2-ethyl-6-methyl-N-(ethoxymethyl)-acetanilide (EMEMA), N-(2-methyl-6-ethylphenyl)-acetamide (MEPA), N-2-ethylphenyl acetamide and 2-ethyl-N-carboxyl aniline were identified during acetochlor degradation, and an anaerobic catabolic pathway of acetochlor was proposed. The toxicity of EMEMA and EMPA for zebrafish, Arabidopsis and Chlorella ellipsoidea were obviously lower than that of acetochlor, indicating that the anaerobic degradation of acetochlor by isolate BAD-10 T is a detoxification process. The work reveals the anaerobic degradation kinetics and catabolic pathway of CAAHs and highlights a potential application of Proteiniclasticum sediminis BAD-10 T for bioremediation of CAAHs residue-contaminated environment., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The positron emission tomography (PET) radiotracer [ 11 C]PBR28 has been increasingly used to image the translocator protein (TSPO) as a marker of neuroinflammation in a variety of brain disorders. Interrelatedly, similar clinical populations can also exhibit altered brain perfusion, as has been shown using arterial spin labelling in magnetic resonance imaging (MRI) studies. Hence, an unsolved debate has revolved around whether changes in perfusion could alter delivery, uptake, or washout of the radiotracer [ 11 C]PBR28, and thereby influence outcome measures that affect interpretation of TSPO upregulation. In this simultaneous PET/MRI study, we demonstrate that [ 11 C]PBR28 signal elevations in chronic low back pain patients are not accompanied, in the same regions, by increases in cerebral blood flow (CBF) compared to healthy controls, and that areas of marginal hypoperfusion are not accompanied by decreases in [ 11 C]PBR28 signal. In non-human primates, we show that hypercapnia-induced increases in CBF during radiotracer delivery or washout do not alter [ 11 C]PBR28 outcome measures. The combined results from two methodologically distinct experiments provide support from human data and direct experimental evidence from non-human primates that changes in CBF do not influence outcome measures reported by [ 11 C]PBR28 PET imaging studies and corresponding interpretations of the biological meaning of TSPO upregulation.

A series of aryl phenoxy methyl triazole conjugated with thiosemicarbazides were designed, synthesized, and evaluated for their tyrosinase inhibitory activities in the presence of l-dopa and l-tyrosine as substrates. All the compounds showed tyrosinase inhibition in the sub-micromolar concentration. Among the derivatives, compound 9j bearing benzyl displayed exceptionally high potency against tyrosinase with IC 50 value of 0.11 μM and 0.17 μM in the presence of l-tyrosine and l-dopa as substrates which is significantly lower than that of kojic acid as the positive control with an IC 50 value of 9.28 μM for l-tyrosine and 9.30 μM for l-dopa. According to Lineweaver-Burk plot, 9j demonstrated an uncompetitive type of inhibition in the kinetic assay. Also, in vitro antioxidant activities determined by DPPH assay recorded an IC 50 value of 68.43 μM for 9i. The melanin content of 9j was determined on B16F10 melanoma human cells which demonstrated a significant reduction of the melanin content. Moreover, the binding energies corresponding to the same ligand as well as computer-aided drug-likeness and pharmacokinetic studies were also carried out. Compound 9j also possessed metal chelation potential correlated to its high anti-TYR activity., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The toxicity of chloroacetamide herbicide in embryo development remains unclear. Acetochlor (AC) is a chloroacetamide that metabolizes into 2‑ethyl‑6‑methyl-2-chloroacetanilide (CMEPA) and 6‑ethyl‑o‑toluidine (MEA). The present study determined the potential effect of AC and its metabolites on embryo development. Both HepG2 cells and zebrafish embryos were exposed to AC, CMEPA and MEA in the presence or absence of co‑treatment with anti‑reactive oxygen species (ROS) reagent N‑acetylcysteine. The generation of ROS, levels of superoxide dismutase (SOD) and glutathione (GSH) in HepG2 cells and lactate dehydrogenase (LDH) leakage from HepG2 cells were investigated. The effects of AC, CMEPA and MEA on DNA breakage, MAPK/ERK pathway activity, viability and apoptosis of HepG2 cells were examined by comet assay, western blotting, MTT assay and flow cytometry, respectively. Levels of LDH, SOD and GSH in zebrafish embryos exposed to AC, CMEPA and MEA were measured. The hatching and survival rates of zebrafish embryos exposed to AC, CMEPA and MEA, were determined, and apoptosis of hatched fish was investigated using acridine orange staining. The present data showed AC, CMEPA and MEA induced generation of ROS and decreased levels of SOD and GSH in HepG2 cells, which in turn promoted DNA breakage and LDH leakage from cells, ultimately inhibiting cell viability and inducing apoptosis, as well as phosphorylation of JNK and P38. However, co‑treatment with N‑acetylcysteine alleviated the pro‑apoptosis effect of AC and its metabolites. Moreover, exposure to AC, CMEPA and MEA lead to toxicity of zebrafish embryos with decreased SOD and GSH and increased LDH levels and cell apoptosis, ultimately decreasing the hatching and survival rates of zebrafish, all of which was attenuated by treatment with N‑acetylcysteine. Therefore, AC and its metabolites (CMEPA and MEA) showed cytotoxicity and embryo development toxicity.

A number of plant-associated proteobacteria have LuxR family transcription factors that we refer to as PipR subfamily members. PipR proteins play roles in interactions between bacteria and their plant hosts, and some are important for bacterial virulence of plants. We identified an ethanolamine derivative, N -(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), as a potent effector of PipR-mediated gene regulation in the plant endophyte Pseudomonas GM79. HEHEAA-dependent PipR activity requires an ATP-binding cassette-type active transport system, and the periplasmic substrate-binding protein (SBP) of that system binds HEHEAA. To begin to understand the molecular basis of PipR system responses to plant factors we crystallized a HEHEAA-responsive SBP in the free- and HEHEAA-bound forms. The SBP, which is similar to peptide-binding SBPs, was in a closed conformation. A narrow cavity at the interface of its two lobes is wide enough to bind HEHEAA, but it cannot accommodate peptides with side chains. The polar atoms of HEHEAA are recognized by hydrogen-bonding interactions, and additional SBP residues contribute to the binding site. This binding mode was confirmed by a structure-based mutational analysis. We also show that a closely related SBP from the plant pathogen Pseudomonas syringae pv tomato DC3000 does not recognize HEHEAA. However, a single amino acid substitution in the presumed effector-binding pocket of the P. syringae SBP converted it to a weak HEHEAA-binding protein. The P. syringae PipR depends on a plant effector for activity, and our findings imply that different PipR-associated SBPs bind different effectors., Competing Interests: The authors declare no competing interest.

Background: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown., Results: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1 °C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCD B2 ), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthR B2 ), a ferredoxin reductase (EthA B2 ), a cytochrome P450 monooxygenase (EthB B2 ), a ferredoxin (EthC B2 ) and a 10-kDa protein of unknown function (EthD B2 ). Complementation with EthABCD B2 and EthABD B2 , but not EthABC B2 in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCD B2 was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCD B2 or EthABD B2 but not EthABC B2 catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthD B2 acted as a ferredoxin in strain B2. EthABD B2 displayed maximal activity at 30 °C and pH 7.5., Conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.

N-methyl-D-aspartate receptors (NMDARs) play critical roles in the physiological function of the mammalian central nervous system (CNS), including learning, memory, and synaptic plasticity, through modulating excitatory neurotransmission. Attributed to etiopathology of various CNS disorders and neurodegenerative diseases, GluN2B is one of the most well-studied subtypes in preclinical and clinical studies on NMDARs. Herein, we report the synthesis and preclinical evaluation of two 11 C-labeled GluN2B-selective negative allosteric modulators (NAMs) containing N,N-dimethyl-2-(1H-pyrrolo[3,2-b]pyridin-1-yl)acetamides for positron emission tomography (PET) imaging. Two PET ligands, namely [ 11 C]31 and [ 11 C]37 (also called N2B-1810 and N2B-1903, respectively) were labeled with [ 11 C]CH 3 I in good radiochemical yields (decay-corrected 28% and 32% relative to starting [ 11 C]CO 2 , respectively), high radiochemical purity (>99%) and high molar activity (>74 GBq/μmol). In particular, PET ligand [ 11 C]31 demonstrated moderate specific binding to GluN2B subtype by in vitro autoradiography studies. However, because in vivo PET imaging studies showed limited brain uptake of [ 11 C]31 (up to 0.5 SUV), further medicinal chemistry and ADME optimization are necessary for this chemotype attributed to low binding specificity and rapid metabolism in vivo.

Hepatic metabolism of low-clearance compound TAK-041 was studied in two different in vitro model systems using rat, dog, monkey, and human suspended cryopreserved hepatocytes and HepatoPac micropatterned coculture model primary hepatocytes. The aim of this work was to investigate the most appropriate system to assess the biotransformation of TAK-041, determine any notable species difference in the rate and in the extent of its metabolic pathways, and establish correlation with in vivo metabolism. TAK-041 exhibited very low turnover in suspended cryopreserved hepatocyte suspensions for all species, with no metabolites observed in human hepatocytes. However, incubations conducted for up to 14 days in the HepatoPac model resulted in more robust metabolic turnover. The major biotransformation pathways of TAK-041 proceed via hydroxylation on the benzene ring fused to the oxotriazine moiety and subsequent sulfate, glucuronide, and glutathione conjugation reactions. The glutathione conjugate of TAK-041 undergoes further downstream metabolism to produce the cysteine S-conjugate, which then undergoes N -acetylation to mercapturic acid and/or conversion to β -lyase-derived thiol metabolites. The minor biotransformation pathways include novel ring closure and hydrolysis, hydroxylation, oxidative N -dealkylation, and subsequent reduction. The HepatoPac model shows a notable species difference in the rate and in the extent of metabolic pathways of TAK-041, with dogs having the fastest metabolic clearance and humans the slowest. Furthermore, the model shows its suitability for establishing correlation with in vivo metabolism of low-turnover and extensively metabolized compounds such as TAK-041, displaying an extensive and unusual downstream sequential β -lyase-derived thiol metabolism in preclinical species and human. SIGNIFICANCE STATEMENT: This study investigated the most appropriate in vitro system to assess the biotransformation of the low-turnover and extensively metabolized compound TAK-041, determine any notable species difference in the rate and in the extent of its metabolic pathways, and establish correlation with in vivo metabolism. The HepatoPac model was identified and showed its suitability for species comparison and establishing correlation, with in vivo metabolism displaying an extensive and unusual downstream sequential β -lyase-derived thiol metabolism in preclinical species and human., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Neuroinflammation is associated with various chronic neurological diseases, including epilepsy; however, neuroimaging approaches for visualizing neuroinflammation have not been used in the clinical routine yet. In this study, we used the translocator protein positron emission tomography (PET) with [ 11 C] DPA713 to investigate neuroinflammation in the epileptogenic zone in patients with child-onset focal epilepsy., Methods: Patients with intractable focal epilepsy were recruited at the Epilepsy Center of Osaka University; those who were taking any immunosuppressants or steroids were excluded. PET images were acquired for 60 min after intravenous administration of [ 11 C] DPA713. The PET image of [ 11 C] DPA713 was co-registered to individual's magnetic resonance imaging (MRI), and the standardized uptake value ratio (SUVr) in regions of interest, which were created in non-lesions and lesions, was calculated using the cerebellum as a pseudo-reference region. In the case of epilepsy surgery, the correlation between SUVr in lesions and pathological findings was analyzed., Results: Twenty-seven patients (mean age: 11.3 ± 6.2 years, male/female: 17/10) were included in this study. Of these, 85.1% showed increased uptake of [ 11 C] DPA713 in the focal epileptic lesion. Three patients showed epileptic spasms, suggesting partial seizure onset, and all 18 patients with abnormal lesions on MRI were similarly highlighted by significant uptake of [ 11 C] DPA713. DPA713-positive patients had a broad range of etiologies, including focal cortical dysplasia, tumors, infarction, and hippocampal sclerosis. Five out of nine MRI-negative patients showed abnormal [ 11 C] DPA713 uptake. The SUVr of [ 11 C] DPA713 in lesions was significantly higher than that in non-lesions. In seven patients who underwent epilepsy surgery, increased [ 11 C] DPA713 uptake was associated with microglial activation., Conclusions: This study indicates that [ 11 C] DPA713 uptake has valuable sensitivity in the identification of epileptic foci in child-onset focal epilepsy, and inflammation is implicated in the pathophysiology in the epileptic foci caused by various etiologies. Further research is required to establish diagnostic tools for identifying focal epileptogenic zones.

Background & Aims: Aryl hydrocarbon receptor (AhR) is a liver-enriched xenobiotic receptor that plays important role in detoxification response in liver. This study aimed to investigate how AhR signaling may impact the pathogenesis of alcohol-related liver disease (ALD)., Methods: Chronic alcohol feeding animal studies were conducted with mouse models of hepatocyte-specific AhR knockout (AhR Δhep ) and NAD(P)H quinone dehydrogenase 1 (NQO1) overexpression, and dietary supplementation of the AhR ligand indole-3-carbinol. Cell studies were conducted to define the causal role of AhR and NQO1 in regulation of redox balance and apoptosis., Results: Chronic alcohol consumption induced AhR activation and nuclear enrichment of NQO1 in hepatocytes of both alcoholic hepatitis patients and ALD mice. AhR deficiency exacerbated alcohol-induced liver injury, along with reduction of NQO1. Consistently, in vitro studies demonstrated that NQO1 expression was dependent on AhR. However, alcohol-induced NQO1 nuclear translocation was triggered by decreased cellular oxidized nicotinamide adenine dinucleotide (NAD + )-to-NADH ratio, rather than by AhR activation. Furthermore, both in vitro and in vivo overexpression NQO1 prevented alcohol-induced hepatic NAD + depletion, thereby enhancing activities of NAD + -dependent enzymes and reversing alcohol-induced liver injury. In addition, therapeutic targeting of AhR in the liver with dietary indole-3-carbinol supplementation efficiently reversed alcoholic liver injury by AhR-NQO1 signaling activation., Conclusions: This study demonstrated that AhR activation is a protective response to counteract alcohol-induced hepatic NAD + depletion through induction of NQO1, and targeting the hepatic AhR-NQO1 pathway may serve as a novel therapeutic approach for ALD., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Polyphenolic compounds (including flavonoids, chalcones, phenolic acids, and furanocoumarins) represent a common part of our diet, but are also the active ingredients of several dietary supplements and/or medications. These compounds undergo extensive metabolism by human biotransformation enzymes and the microbial flora of the colon. CYP2D6 enzyme metabolizes approximately 25% of the drugs, some of which has narrow therapeutic window. Therefore, its inhibition can lead to the development of pharmacokinetic interactions and the disruption of drug therapy. In this study, the inhibitory effects of 17 plant-derived compounds and 19 colonic flavonoid metabolites on CYP2D6 were examined, employing two assays with different test substrates. The O-demethylation of dextromethorphan was tested employing CypExpress 2D6 kit coupled to HPLC analysis; while the O-demethylation of another CYP2D6 specific substrate (AMMC) was investigated in a plate reader assay with BioVision Fluorometric CYP2D6 kit. Interestingly, some compounds (e.g., bergamottin) inhibited both dextromethorphan and AMMC demethylation; however, certain substances proved to be inhibitors only in one of the assays applied. Our results demonstrate that some polyphenols and colonic metabolites are inhibitors of CYP2D6-catalyzed reactions. Nevertheless, the inhibitory effects showed strong substrate dependence., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Objective: Despite combined antiretroviral therapy, neuroinflammation may persist in persons living with HIV (PLWH) and contribute to cognitive impairment in this population. Positron emission tomography (PET) imaging targeting 18 kDa translocator protein (TSPO) has been used to localize neuroinflammation. We aimed to use TSPO-PET imaging to evaluate neuroinflammation in PLWH., Design: Twenty-four virologically suppressed PLWH on combined antiretroviral therapy and 13 HIV-negative (HIV-) controls completed TSPO-PET imaging using the radiotracer [C]PBR28. Because of tracer complexity and differing procedures used in previous studies, we employed an expansive methodological approach, using binding potential (BP) and standard uptake value ratio and multiple different reference regions to estimate [C]PBR28 binding., Methods: [C]PBR28 binding was measured in 30 cortical and subcortical regions and compared between PLWH and HIV- controls. Pearson correlation evaluated the association between [C]PBR28 binding and cognition and clinical measures of HIV., Results: Analyses conducted using multiple reference regions and measures of tracer uptake revealed no significant differences between [C]PBR28 binding in PLWH compared with HIV- controls. In addition, [C]PBR28 binding in PLWH was not significantly associated with clinical measures of HIV or plasma biomarkers of inflammation. [C]PBR28 binding was not significantly elevated in cognitively impaired PLWH compared with unimpaired PLWH, but there were inverse relationships between cognitive performance (executive and global function) and [C]PBR28 binding in PLWH., Conclusions: Our results suggest that neuroinflammation may play a role in cognitive deficits, but overall neuroinflammatory levels as measured by TSPO-PET imaging in PLWH are not significantly different from those seen in HIV- controls.

Gefitinib and erlotinib are epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) with activity against metastatic non-small cell lung cancer. Aldehyde oxidase-1 (AOX1) is a cytosolic drug-metabolizing enzyme. We conducted an experimental and molecular docking study on the effect of gefitinib, erlotinib, and select metabolites on the in vitro catalytic activity of AOX1, as assessed by carbazeran 4-oxidation, and determined the impact of AOX1 inhibition on hepatic metabolism of zaleplon and methotrexate. Gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib inhibited human hepatic cytosolic carbazeran 4-oxidation by a competitive mode, with inhibition constants in submicromolar or low micromolar concentrations. Desmethylgefitinib did not affect AOX1 catalytic activity. A similar pattern was obtained when investigated with human kidney cytosol or recombinant AOX1. The differential effect of gefitinib on human, rat, and mouse hepatic AOX1 catalytic activity suggests species-dependent chemical inhibition of AOX1. Erlotinib was considerably more potent than gefitinib in decreasing hepatic cytosolic zaleplon 5-oxidation and methotrexate 7-oxidation. Molecular docking analyses provided structural insights into the interaction between EGFR-TKIs and AOX1, with key residues and bonds identified, which provided favorable comparison and ranking of potential inhibitors. Based on the US Food and Drug Administration guidance to assess the risk of drug-drug interactions, the calculated R 1 values indicate that further investigations are warranted to determine whether gefitinib and erlotinib impact AOX1-mediated drug metabolism in vivo. Overall, erlotinib desmethylerlotinib, didesmethylerlotinib, gefitinib, and desmorpholinopropylgefitinib are potent inhibitors of human AOX1 catalytic function and hepatic metabolism of zaleplon and methotrexate, potentially affecting drug efficacy or toxicity. SIGNIFICANCE STATEMENT: As epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs), gefitinib and erlotinib are first-line pharmacotherapy for metastatic non-small cell lung cancer. Our experimental findings indicate that clinically relevant concentrations of gefitinib, desmorpholinopropylgefitinib, erlotinib, desmethylerlotinib, and didesmethylerlotinib, but not desmethylgefitinib, inhibit human aldehyde oxidase (AOX1) catalytic activity and hepatic cytosolic metabolism of zaleplon and methotrexate. Molecular docking analysis provide structural insights into the key AOX1 interactions with these EGFR-TKIs. Our findings may trigger improved strategies for new EGFR-TKI design and development., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Chlorine dioxide has been reported as very efficiently removing pesticides and other organic compounds from water matrixes. Due to pesticide toxicity and potential toxicity of their degradation products, it is important to monitor these compounds as environmental pollutants in ground and surface waters. Evaluating the effects of chlorine dioxide treatment is necessary, and toxicity studies are used to ascertain the severity of effects of intermediates due to incomplete degradation of the parent compounds. In this paper, for the first time, chlorine dioxide is applied and evaluated for the removal of chloroacetamide herbicides (pethoxamid and metazachlor) from waters (deionized water and Sava River water). The degradation degree of herbicides was measured by high-performance liquid chromatography, the main degradation products were identified using gas chromatography with a triple quadrupole mass detector, and the degree of mineralization was monitored by total organic carbon analysis. Four and two degradation products were identified after pethoxamid and metazachlor degradation, respectively. Total organic carbon analysis showed mineralization occurred, but it was incomplete. The mineralization and the characteristics of the degradation products obtained were tested using Daphnia magna and showed lower toxicity than the parent herbicides. The advantage of the applied treatment was a very high degradation percentage for pethoxamid removal from deionized water and Sava River water (100% and 97%, respectively), with higher mineralization efficiency (65%) than metazachlor. Slightly lower degradation efficiency in the Sava River water was due to chlorine dioxide oxidizing the herbicides and dissolved organic matter simultaneously.

Herbicides and their metabolites are often detected in water bodies where they may cause adverse effects to non-target organisms. Their effects at environmentally relevant concentrations are often unclear, especially concerning mixtures of pesticides. This study thus investigated the impacts of one of the most used herbicides: S-metolachlor and its two metabolites, metolachlor oxanilic acid (MOA) and metolachlor ethanesulfonic acid (MESA) on the development of zebrafish embryos (Danio rerio). Embryos were exposed to the individual substances and their environmentally relevant mixture until 120 hpf (hours post-fertilization). The focus was set on sublethal endpoints such as malformations, hatching success, length of fish larvae, spontaneous movements, heart rate and locomotion. Moreover, expression levels of eight genes linked to the thyroid system disruption, oxidative stress defense, mitochondrial metabolism, regulation of cell cycle and retinoic acid (RA) signaling pathway were analyzed. Exposure to S-metolachlor (1 μg/L) and the pesticide mixture (1 μg/L of each substance) significantly reduced spontaneous tail movements of 21 hpf embryos. Few rare developmental malformations were observed, but only in larvae exposed to more than 100 μg/L of individual substances (craniofacial deformation, non-inflated gas bladder, yolk sac malabsorption) and to 30 μg/L of each substance in the pesticide mixture (spine deformation). No effect on hatching success, length of larvae, heart rate or larvae locomotion were found. Strong responses were detected at the molecular level including induction of p53 gene regulating the cell cycle (the pesticide mixture - 1 μg/L of each substance; MESA 30 μg/L; and MOA 100 μg/L), as induction of cyp26a1 gene encoding cytochrome P450 (pesticide mixture - 1 μg/L of each substance). Genes implicated in the thyroid system regulation (dio2, thra, thrb) were all overexpressed by the environmentally relevant concentrations of the pesticide mixture (1 μg/L of each substance) and MESA metabolite (1 μg/L). Zebrafish thyroid system disruption was revealed by the overexpressed genes, as well as by some related developmental malformations (mainly gas bladder and yolk sac abnormalities), and reduced spontaneous tail movements. Thus, the thyroid system disruption represents a likely hypothesis behind the effects caused by the low environmental concentrations of S-metolachlor, its two metabolites and their mixture., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Transient receptor potential channel subfamily A member 1 (TRPA1) is a Ca 2+ -permeable cation channel that serves as one of the primary sensors of environmental irritants and noxious substances. Many TRPA1 agonists are electrophiles that are recognized by TRPA1 via covalent bond modifications of specific cysteine residues located in the cytoplasmic domains. However, a mechanistic understanding of electrophile sensing by TRPA1 has been limited due to a lack of high-resolution structural information. Here, we present the cryoelectron microscopy (cryo-EM) structures of nanodisc-reconstituted ligand-free TRPA1 and TRPA1 in complex with the covalent agonists JT010 and BITC at 2.8, 2.9, and 3.1 Å, respectively. Our structural and functional studies provide the molecular basis for electrophile recognition by the extraordinarily reactive C621 in TRPA1 and mechanistic insights into electrophile-dependent conformational changes in TRPA1. This work also provides a platform for future drug development targeting TRPA1., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The study concerned dissipation of metazachlor and clomazone, herbicides widely used in rapeseed (Brassica napus L. subsp. napus) protection, applied to the clay soil under field and laboratory conditions. Furthermore, the uptake of these pesticide from soil by rapeseed plants was investigated under field conditions. An additional aim of this work was to modify the QuEChERS method for the determination of metazachlor and clomazone in the plant material. Analytical procedures for metazachlor and clomazone qualification and quantification in rapeseed plants and soil were developed, using gas chromatography with an micro electron capture detector (GC-μECD) and a mass detector (GC-MS/MS QqQ) as confirmation. Dissipation kinetics of herbicide residues in soil were described as first-order equations. The analytical performance was very satisfactory and confirmed that the methods meet the requirements of the European Commission. In the conducted field experiments it was found that dissipation of clomazone and metazachlor in clay soil follows first-order kinetics (R 2 between 0.964 and 0.978), and half-lives were 9.5 days and 10.2 days for clomazone and metazachlor, respectively. Under laboratory conditions, dissipation of clomazone and metazachlor in soil also follows first-order kinetics (R 2 between 0.937 and 0.938), and half-lives were 8.8 days and 5.7 days for clomazone and metazachlor, respectively. Residues of both herbicides in rape plants 22 days after application of herbicides were below the maximum residue levels for Brassica plants. Metazachlor and clomazone dissipate very fast in clay soil and their uptake by rape plants is very low.

Background: Yarrowia lipolytica is an oleaginous yeast that can be genetically engineered to produce lipid and non-lipid biochemicals from a variety of feedstocks. Metabolic engineering of this organism usually requires genetic markers in order to select for modified cells. The potential to combine multiple genetic manipulations depends on the availability of multiple or recyclable selectable markers., Results: We found that Y. lipolytica has the ability to utilize acetamide as the sole nitrogen source suggesting that the genome contains an acetamidase gene. Two potential Y. lipolytica acetamidase gene candidates were identified by homology to the A. nidulans acetamidase amdS. These genes were deleted in the wild-type Y. lipolytica strain YB-392, and deletion strains were evaluated for acetamide utilization. One deletion strain was unable to grow on acetamide and a putative acetamidase gene YlAMD1 was identified. Transformation of YlAMD1 followed by selection on acetamide media and counterselection on fluoroacetamide media showed that YlAMD1 can be used as a recyclable genetic marker in Saccharomyces cerevisiae and Ylamd1Δ Y. lipolytica., Conclusions: These findings add to our understanding of Y. lipolytica nitrogen utilization and expand the set of genetic tools available for engineering this organism, as well as S. cerevisiae.

Synergistic biodegradation of earthworms and soil microorganisms plays a key role in the removal of organic pollutants in soil, yet microbially mediated processes remain unclear, especially regarding the succession of soil microbial interactions. Herein, soil biochemical evaluation, microbial community characterization, and interaction network construction were combined to understand the mechanisms dominating microbial community succession during synergistic bioremediation of metolachlor-polluted soils. The results of the network analysis indicated that metolachlor could render more complex relations but weaker connection strength among soil microorganisms. The addition of earthworms significantly alleviated the stress of metolachlor on soil microbial interactions and resulted in the restoration of interactions to a great extent. Additionally, the soil physicochemical properties, enzyme activities, and microbial community changed greatly with the addition of metolachlor and earthworms. Some soil microorganisms became significantly correlated with soil properties, metolachlor concentrations, and enzyme activities. These results, dominated by the succession of soil microbial communities, provide a new perspective for assessing the remediation effect of contaminated soil by organic pollutants., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The effects of the herbicide metazachlor and its major metabolite metazachlor OA at two concentrations, including environmentally relevant concentrations of metazachlor (0.0115 µmol/l and 0.0790 µmol/l) and metazachlor OA (0.0117 µmol/l and 0.0805 µmol/l), respectively, were evaluated on early ontogeny, growth, behaviour, oxidative stress, antioxidant enzyme levels, histology, and mortality of marbled crayfish Procambarus virginalis. Both tested concentrations of metazachlor and metazachlor OA were associated with significantly lower growth and delayed ontogenetic development compared to controls. Exposure of metazachlor at 0.0115 µmol/l and metazachlor OA at 0.0117 µmol/l and 0.0805 µmol/l resulted in significantly lower activity of total superoxide dismutase (SOD), catalase (CAT), glutathione s-transferase (GST), glutathione reductase (GR), and reduced glutathione (GSH) compared with control and resulted in gill anomalies ranging from wall thinning to focal disintegration of branchial structure. Metazachlor at the environmentally relevant concentration of 0.0790 µmol/l was associated with significant alterations of crayfish distance moved and walking speed. The potential risk associated with metazachlor use in agriculture related to effects on non-target aquatic organisms.

Oxidative stress-induced neuroinflammation is the prominent feature of neurodegenerative disorders, and is characterized by a gradual decline of structure and function of neurons. Many biochemical events emerge thanks to the result of this neurodegeneration, and ultimately provoke neuroinflammation, activation of microglia, and oxidative stress, leading to neuronal death. This cascade not only explains the complexity of events taking place across different stages, but also depicts the need for more effective therapeutic agents. The present study was designed to investigate the neuroprotective effects of newly synthesized benzimidazole containing acetamide derivatives, 3a (2-(4-methoxyanilino)-N-[1-(4-methylbenzene-1-sulfonyl)-1H-benzimidazol-2-yl] acetamide) and 3b (2-(Dodecylamino)-N-[1-(4-methylbenzene-1-sulfonyl)-1H-benzimidazol-2-yl] acetamide) against ethanol-induced neurodegeneration in the rat model. Both derivatives were characterized spectroscopically by proton NMR ( 1 H-NMR) and carbon-13 NMR ( 13 C-NMR) and evaluated for neuroprotective potential using different pharmacological approaches. In vivo experiments demonstrated that ethanol triggered neurodegeneration characterized by impaired antioxidant enzymes and elevated oxidative stress. Furthermore, ethanol administration induced neuroinflammation, as demonstrated by elevated expression of tumor necrotic factor (TNF-α), nuclear factor κB (NF-κB), cyclooxygenase-2 (COX2), and ionized calcium-binding adapter molecule-1 (Iba-1), which was further validated by enzyme-linked immunosorbent assay (ELISA). Treatment with 3a and 3b ameliorated the ethanol-induced oxidative stress, neuroinflammation, and memory impairment. The affinity of synthesized derivatives towards various receptors involved in neurodegeneration was assessed through docking analysis. The versatile nature of benzimidazole nucleus and its affinity toward several receptors suggested that it could be a multistep targeting neuroprotectant. As repetitive clinical trials of neuroprotectants targeting a single step of the pathological process have failed previously, our results suggested that a neuroprotective strategy of acting at different stages may be more advantageous to intervene in the vicious cycles of neuroinflammation.

Fragment-based approach combined with electrophilic reactive compounds is a powerful strategy to discover novel covalent ligands for protein target. However, the promiscuous reactivity often interferes with identification of the fragments possessing specific binding affinity to the targeted protein. In our study, we report the fragment-based covalent drug discovery using the chemically tuned weak reactivity of chlorofluoroacetamide (CFA). We constructed a small fragment library composed of 30 CFA-appended compounds and applied it to the covalent ligand screening for cysteine protease papain as a model protein target. Using the fluorescence enzymatic assay, we identified CFA-benzothiazole 30 as a papain inhibitor, which was found to irreversibly inactivate papain upon enzyme kinetic analysis. The formation of the covalent papain-30 adduct was confirmed using electrospray ionization mass spectrometry analysis. The activity-based protein profiling (ABPP) experiment using an alkynylated analog of 30 (i.e., 30-yne) revealed that 30-yne covalently labeled papain with high selectivity. These data demonstrate potential utility of the CFA-fragment library for de novo discovery of target selective covalent inhibitors.

Wasabi (Eutrema japonicum) is a perennial plant native to Japan that is used as a spice because it contains isothiocyanates. It also contains an isosaponarin, 4'-O-glucosyl-6-C-glucosyl apigenin, in its leaves, which has received increasing attention in recent years for its bioactivity, such as its promotion of type-I collagen production. However, its biosynthetic enzymes have not been clarified. In this study, we partially purified a C-glucosyltransferase (CGT) involved in isosaponarin biosynthesis from wasabi leaves and identified the gene coding for it (WjGT1). The encoded protein was similar to UGT84 enzymes and was named UGT84A57. The recombinant enzyme of WjGT1 expressed in Escherichia coli showed C-glucosylation activity toward the 6-position of flavones such as apigenin and luteolin. The enzyme also showed significant activity toward flavonols, but trace or no activity toward flavone 4'-O-glucosides, suggesting that isosaponarin biosynthesis in wasabi plants would proceed by 6-C-glucosylation of apigenin, followed by its 4'-O-glucosylation. Interestingly, the enzyme showed no activity against sinapic acid or p-coumaric acid, which are usually the main substrates of UGT84 enzymes. The accumulation of WjGT1 transcripts was observed mainly in the leaves and flowers of wasabi, in which C-glucosylflavones were accumulated. Molecular phylogenetic analysis suggested that WjGT1 acquired C-glycosylation activity independently from other reported CGTs after the differentiation of the family Brassicaceae., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

To investigate the induction of oxidative stress and antioxidant response in the chloroacetanilides-degrading Trichoderma spp. under alachlor and metolachlor exposure, a comparative analysis using popular biomarkers was employed. An increased intracellular level of reactive oxygen species (ROS; especially superoxide anion [O 2 - ]) as well as products of lipid and protein oxidation after 24 h incubation with the herbicides confirmed chloroacetanilide-induced oxidative stress in tested Trichoderma strains. However, the considerable decline in the ROS levels and the carbonyl group content (biomarkers of protein peroxidation) in a time-dependent manner and changes in the antioxidant enzyme activities indicated an active response against chloroacetanilide-induced oxidative stress and the mechanism of tolerance in tested fungi. Moreover, the tested herbicides clearly modified the phospholipids (PLs) content in Trichoderma spp. in the stationary phase of growth, which was manifested through the difference in phosphatidic acid (PA), phosphatidylethanolamine (PE) and phosphatidylcholines (PC) levels. Despite enhanced lipid peroxidation and changes in PLs in most tested fungi, only a slight modification in membrane integrity of Trichoderma spp. under chloroacetanilides exposure was noted. The obtained results suggest that the alterations in the antioxidant system and the PLs profile of Trichoderma spp. might be useful biomarkers of chloroacetanilide-induced oxidative stress., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Increasing production of energy crops in Europe, mainly maize and rapeseed, has altered patterns of pesticide use in recent decades. The long-term effects of S-metolachlor (S-M) and of its metabolite metolachlor OA (M-OA) at the environmentally relevant concentration of 4.2 μg L -1 and at 42 μg L -1 (ten-fold concentration) on marbled crayfish (Procambarus virginalis) were evaluated in a 28-day exposure and after a subsequent 28-day recovery period. Indicators assessed were behaviour; biochemical haemolymph profile; oxidative and antioxidant parameters of gill, hepatopancreas, and muscle; and histology of hepatopancreas and gill. Results showed biochemical haemolymph profile (lactate, alanine aminotransferase, aspartate aminotransferase, inorganic phosphate), lipid peroxidation in hepatopancreas, and antioxidant parameters (catalase, reduced glutathione, glutathione S-transferase) of hepatopancreas and gill of crayfish exposed to S-M and M-OA to significantly differ from controls (P < 0.01). Antioxidant biomarker levels remained different from controls after a 28-day recovery period. Differences in behaviour including speed of movement and velocity, and histopathological damage to gill and hepatopancreas were associated with S-M and M-OA exposure and persisted after 28 days in S-M- and M-OA-free water. Results suggest harmful effects of low concentrations of S-M and its metabolite M-OA on non-target organisms and provide information for assessing their effects at environmentally relevant concentrations., (Copyright © 2019. Published by Elsevier Ltd.)

Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-O 6 -benzylguanine, N-(2-dimethylamino)ethyl)acridine-4-carboxamide, zaleplon, phthalazine, BIBX1382 [ N 8-(3-Chloro-4-fluorophenyl)- N 2-(1-methyl-4-piperidinyl)-pyrimido[5,4- d ]pyrimidine-2,8-diamine dihydrochloride], and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms 1 by synchronization to environmental cues and is involved in diverse physiological processes 2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function 3 . Melatonin is formed in the pineal gland in a light-regulated manner 4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness 5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT 1 ) and type 1B (MT 2 ) 3,6 . Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden 7 . Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids 8,9 , and is one of the most popular supplements in the United States 10 . Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT 1 in complex with four agonists: the insomnia drug ramelteon 11 , two melatonin analogues, and the mixed melatonin-serotonin antidepressant agomelatine 12,13 . The structure of MT 2 is described in an accompanying paper 14 . Although the MT 1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT 1 , access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT 1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors.

Degradation of metolachlor in surface soil is extremely important to its potential mobility and overall persistence. In this study, the effects of earthworms (Eisenia fetida) on the degradation of metolachlor at two concentration levels (5 and 20 mg kg -1 ) in soil were investigated via the column experiment. The degradation kinetics of metolachlor indicate that addition of earthworms enhances metolachlor degradation significantly (P < 0.05), with the enhanced degradation rate of 30% and 63% in the low and high concentration treatments at the 15th day, respectively. Fungi rather than bacteria are primarily responsible for metolachlor degradation in soil, and earthworms stimulate metolachlor degradation mainly by stimulating the metolachlor-degrading functional microorganisms and improving fungal community structure. Earthworms prefer to promote the possible fungal degraders like order Sordariales, Microascales, Hypocreales and Mortierellales and the possible bacteria genus Rubritalea and strengthen the relationships between these primary fungi. Two metabolites metolachlor oxanilic (MOXA) and moetolachlor ethanesulfonic acid (MESA) are detected in soil and earthworms in the high concentration treatments. Earthworms stimulate the formation of MOXA and yet inhibit the formation of MESA in soil. Another metabolite metolachlor-2-hydroxy (M2H) is also detected in earthworms, which is reported firstly. The study provides an important information for the remediation of metolachlor-polluted soil., (Copyright © 2019. Published by Elsevier Ltd.)

The filamentous fungus Trichoderma koningii is capable of fast and effective eliminate alachlor (90% after 72 h when added separately and 80-60% in the presence of 1-5 mM of copper). After 168 h over 99% elimination of alachlor resulted in detoxification and was connected with the mitigation of reactive oxygen species (ROS) production. Using MS/MS techniques, seven dechlorinated and hydroxylated metabolites were identified. Cytochrome P450 and laccase participate in biotransformation of the herbicide by this non-ligninolytic fungus. Laccase activity is stimulated both by copper and the mixture of copper and alachlor, which seems to be important for combined pollutants. T. koningii is characterized by high tolerance to copper (up to 7.5 mM). The metal content in mycelia reached 0.9-7.76 mg in 1 g of dry biomass. Our results suggest that T. koningii strain seems to be a promising tool for bioremediation of agricultural areas co-contaminated with copper-based fungicides and chloroacetanilide herbicides., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Described herein is the synthesis and biological evaluation of a series of non-carboxylic inhibitors of Protein Tyrosine Phosphatase 1B designed using bioisosteric replacement strategy. Six N-(3-(1H-tetrazol-5-yl)phenyl)acetamide derivatives designed employing the aforementioned strategy were synthesized and screened for PTP1B inhibitory activity. Among the synthesized compounds, compound NM-03 exhibited the most potent inhibitory activity with IC 50 value of 4.48 µM. Docking studies with NM-03 revealed the key interactions with desired amino acids in the binding site of PTP1B. Furthermore, compound NM-03 also elicited good in vivo activity. Taken together, the results of this study establish N-(3-(1H-tetrazole-5-yl)phenyl)-2-(benzo[d]oxazol-2-ylthio)acetamide (NM-03) as a valuable lead molecule with great potential for PTP1B inhibitor development targeting diabetes., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Certain plant-associated Proteobacteria sense their host environment by detecting an unknown plant signal recognized by a member of a LuxR subfamily of transcription factors. This interkingdom communication is important for both mutualistic and pathogenic interactions. The Populus root endophyte Pseudomonas sp. GM79 possesses such a regulator, named PipR. In a previous study we reported that PipR activates an adjacent gene ( pipA ) coding for a proline iminopeptidase in response to Populus leaf macerates and peptides and that this activation is dependent on a putative ABC-type transporter [Schaefer AL, et al. (2016) mBio 7:e01101-16]. In this study we identify a chemical derived from ethanolamine that induces PipR activity at picomolar concentrations, and we present evidence that this is the active inducer present in plant leaf macerates. First, a screen of more than 750 compounds indicated ethanolamine was a potent inducer for the PipR-sensing system; however, ethanolamine failed to bind to the periplasmic-binding protein (PBP) required for the signal response. This led us to discover that a specific ethanolamine derivative, N -(2-hydroxyethyl)-2-(2-hydroxyethylamino) acetamide (HEHEAA), binds to the PBP and serves as a potent PipR-dependent inducer. We also show that a compound, which coelutes with HEHEAA in HPLC and induces pipA gene expression in a PipR-dependent manner, can be found in Populus leaf macerates. This work sheds light on how plant-associated bacteria can sense their environment and on the nature of inducers for a family of plant-responsive LuxR-like transcription factors found in plant-associated bacteria., Competing Interests: The authors declare no conflict of interest.

The nucleophilic attack of hydrogen sulfide (HS - ) on six different chloroacetanilide herbicides was evaluated theoretically using the dispersion-corrected hybrid functional wB97XD and the 6-311++G(2d,2p) Pople basis sets. The six evaluated substrates were propachlor (A), alachlor (B), metolachlor (C), tioacetanilide (D), β-anilide (E), and methylene (F). Three possible mechanisms were considered: (a) bimolecular nucleophilic substitution (S N ²) reaction mechanism, (b) oxygen assistance, and (c) nitrogen assistance. Mechanisms based on O- and N-assistance were discarded due to a very high activation barrier in comparison with the corresponding S N ² mechanism, with the exception of compound F. The N-assistance mechanism for compound F had a free activation energy of 23.52 kcal/mol, which was close to the value for the corresponding S N ² mechanism (23.94 kcal/mol), as these two mechanisms could occur in parallel reactions with almost 50% of each one. In compounds A to D, an important electron-withdrawing effect of the C=O and C=S groups was seen, and consequently, the activation free energies in these S N ² reactions were smaller, with a value of approximately 18 kcal/mol. Instead, compounds E and F, which have a CH₂ group in the β-position, presented a higher activation free energy (≈22 kcal/mol). Good agreement was found between experimental and theoretical values for all cases, and a reaction force analysis was performed on the intrinsic reaction coordinate profile in order to gain more details about the reaction mechanism. Finally, from the natural bond orbital (NBO) analysis, it was possible to evaluate the electronic reorganization through the reaction pathway where all the transition states were early in nature in the reaction coordinate (δBav < 50%); the transition states corresponding to compounds A to D turned out to be more synchronous than those for compounds E and F.

We present the results of a two years study on the contamination of the Luxembourg Sandstone aquifer by metolachlor-ESA and metolachlor-OXA, two major transformation products of s-metolachlor. The aim of the study was twofold: (i) assess whether elevated concentrations of both transformation products (up to 1000 ng/l) were due to fast flow breakthough events of short duration or the signs of a contamination of the entire aquifer and (ii) estimate the time to trend reversal once the parent compound was withdrawn from the market. These two questions were addressed by a combined use of groundwater monitoring, laboratory experiments and numerical simulations of the fate of the degradation products in the subsurface. Twelve springs were sampled weekly over an eighteen month period, and the degradation rates of both the parent compound and its transformation products were measured on a representative soil in the laboratory using a radiolabeled precursor. Modelling with the numeric code PEARL simulating pesticide fate in soil coupled to a simple transfer function model for the aquifer compartment, and calibrated from the field and laboratory data, predicts a significant damping by the aquifer of the peaks of concentration of both metolachlor-ESA and -OXA leached from the soil. The time to trend reversal following the ban of s-metolachlor in spring protection zones should be observed before the end of the decade, while the return of contaminant concentrations below the drinking water limit of 100 ng/l however is expected to last up to twelve years. The calculated contribution to total water discharge of the fast-flow component from cropland and short-circuiting the aquifer was small in most springs (median of 1.2%), but sufficient to cause additional peaks of concentration of several hundred nanograms per litre in spring water. These peaks are superimposed on the more steady contamination sustained by the base flow, and should cease immediately once application of the parent compound stops., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Myofibroblasts contribute to fibrosis through the overproduction of extracellular matrix (ECM) proteins, primarily type I collagen (COL-1) and fibronectin (FN), a process which is mediated in systemic sclerosis (SSc) by the activation of fibrogenic intracellular signaling transduction molecules, including extracellular signal-regulated kinases 1 and 2 (Erk1/2) and protein kinase B (Akt). Selexipag is a prostacyclin receptor agonist synthesized for the treatment of pulmonary arterial hypertension. The study investigated the possibility for selexipag and its active metabolite (ACT-333679) to downregulate the profibrotic activity in primary cultures of SSc fibroblasts/myofibroblasts and the fibrogenic signaling molecules involved., Methods: Fibroblasts from skin biopsies obtained with Ethics Committee (EC) approval from patients with SSc, after giving signed informed consent, were cultured until the 3 rd culture passage and then either maintained in normal growth medium (untreated cells) or independently treated with different concentrations of selexipag (from 30 μM to 0.3 μM) or ACT-333679 (from 10 μM to 0.1 μM) for 48 h. Protein and gene expressions of α-smooth muscle actin (α-SMA), fibroblast specific protein-1 (S100A4), COL-1, and FN were investigated by western blotting and quantitative real-time PCR. Erk1/2 and Akt phosphorylation was investigated in untreated and ACT-333679-treated cells by western botting., Results: Selexipag and ACT-333679 significantly reduced protein synthesis and gene expression of α-SMA, S100A4, and COL-1 in cultured SSc fibroblasts/myofibroblasts compared to untreated cells, whereas FN was significantly downregulated at the protein level. Interestingly, ACT-333679 significantly reduced the phosphorylation of Erk1/2 and Akt in cultured SSc fibroblasts/myofibroblasts., Conclusions: Selexipag and mainly its active metabolite ACT-333679 were found for the first time to potentially interfere with the profibrotic activity of cultured SSc fibroblasts/myofibroblasts at least in vitro, possibly through the downregulation of fibrogenic Erk1/2 and Akt signaling molecules.

Aim: The aim of this study was to study the effect of celecoxib on agomelatine metabolism in vitro and in vivo., Methods: Ten healthy male Sprague-Dawley rats were randomly divided into 2 groups: Group A (control group) and Group B (30 mg/kg celecoxib). Then a single dose of 20 mg/kg agomelatine was administered orally 30 min after administration of celecoxib. In an in vitro study, celecoxib with a series of concentrations was added to an incubation mixture containing recombinant human CYP2C9, human or rat liver microsomes to determine the half-maximal inhibitory concentration on the metabolism of agomelatine. Moreover, a mechanism study was performed to determine the inhibitory effect of celecoxib on CYP2C9., Results: The results showed that a single dose of 30 mg/kg celecoxib significantly increased the area under the concentration-time curve and maximum concentration of agomelatine. In addition, celecoxib inhibited the metabolism of agomelatine in the in vitro studies, which was determined to be by a competitive mechanism on CYP2C9. Those results indicated that celecoxib has an inhibitory effect on the metabolism of agomelatine both in vivo and in vitro., Conclusion: Thus, more attention should be paid when celecoxib is administered combined with agomelatine., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The objective of present study was to develop a nanoemulsion formulation of agomelatine (BCS class II drug) for the solubility enhancement. Capmul MCM, Tween 80 and PEG-400 were selected as oil, surfactant and co-surfactant respectively. The high energy ultrasonication method was used for the preparation of nanoemulsion. Three-factor three-level central composite design was employed to get the best formulation. The independent variables selected for the optimization were % oil, %S mix and sonication time (second). Based on the constraints applied to independent and dependent variables, the optimized formulation was selected with 2% oil, 10% S mix and 45s sonication time. The experimental values for dependent variables such as hydrodynamic diameter (nm), % transmittance and % CDR were found to be 73.72±2.53nm, 98.2±0.42%, 84.71±4.05% respectively. TEM and AFM-assisted morphological characterization of optimized Ago-NE was done and it was found with a spherical shape. The PDI, Zeta potential and the refractive index of optimized Ago-NE were found to be 0.137±0.016, -7.40±0.12mV and 1.423±0.045 respectively. The viscosity, pH and drug content of optimized Ago-NE were found as 25.12±0.67cP, 6.4±0.17 and 97.83±1.03% respectively. The ex-vivo permeation profile of optimized Ago-NE and agomelatine suspension through goat nasal mucosa were compared till 12h and % cumulative drug permeated was found to be 90% and 40% respectively. The higher drug permeation profile of optimized Ago-NE confirmed that the solubility of agomelatine has been improved., (Copyright © 2017 Elsevier B.V. All rights reserved.)

eIF2B is a dedicated guanine nucleotide exchange factor for eIF2, the GTPase that is essential to initiate mRNA translation. The integrated stress response (ISR) signaling pathway inhibits eIF2B activity, attenuates global protein synthesis and upregulates a set of stress-response proteins. Partial loss-of-function mutations in eIF2B cause a neurodegenerative disorder called Vanishing White Matter Disease (VWMD). Previously, we showed that the small molecule ISRIB is a specific activator of eIF2B (Sidrauski et al., 2015). Here, we report that various VWMD mutations destabilize the decameric eIF2B holoenzyme and impair its enzymatic activity. ISRIB stabilizes VWMD mutant eIF2B in the decameric form and restores the residual catalytic activity to wild-type levels. Moreover, ISRIB blocks activation of the ISR in cells carrying these mutations. As such, ISRIB promises to be an invaluable tool in proof-of-concept studies aiming to ameliorate defects resulting from inappropriate or pathological activation of the ISR., Competing Interests: YW, LL The authors were employees of Calico Life Sciences LLC at the time the study was conducted and have no other competing financial interests to declare. RE The authors were employees of AbbVie at the time the study was conducted and have no other competing financial interests to declare. HL Hock Ben Lim: The authors were employees of AbbVie at the time the study was conducted and have no other competing financial interests to declare. CS Chaohong Sun: The authors were employees of AbbVie at the time the study was conducted and have no other competing financial interests to declare. CS The author was an employee of Calico Life Sciences LLC at the time the study was conducted. The author is listed as an inventor on a patent application describing ISRIB. Rights to the patent application have been licensed to Calico Life Sciences LLC from the University of California, San Francisco. The author has no other competing financial interests to declare., (© 2018, Wong et al.)

Vasopressin 1B receptors (V 1B Rs) are abundantly expressed in the pituitary, and in vivo PET of V 1B Rs was recently enabled by our development of a specific radioligand, 11 C-TASP0434299, derivatized from pyridopyrimidin-4-one. Here, we identified a novel pyridopyrimidin-4-one analog, N - tert -butyl-2-[2-(6- 11 C-methoxypyridine-2-yl)-6-[3-(morpholin-4-yl)propoxy]-4-oxopyrido[2,3- d ]pyrimidin-3(4 H )-yl]acetamide ( 11 C-TASP0410699, hereafter referred to as 11 C-TASP699), as a potent V 1B R radioligand producing a higher image contrast for the target than 11 C-TASP0434299. Methods: In vitro properties of TASP699 were assessed by assaying its affinity for human V 1B R and its selectivity for off-target molecules. Radioactive uptake in the pituitary was analyzed using PET in rhesus monkeys after intravenous administration of 11 C-TASP699. Serial doses of a selective V 1B R antagonist, 2-[2-(3-chloro-4-fluorophenyl)-6-[3-(morpholin-4-yl)propoxy]-4-oxopyrido[2,3- d ]pyrimidin-3(4 H )-yl]- N -isopropylacetamide hydrochloride (TASP0390325), were administered before the radioligand injection. Autoradiographic labeling of monkey pituitary slices with 11 C-TASP699 was conducted with or without nonradioactive V 1B R antagonists. Results: The half maximal inhibitory concentration (IC 50 ) of TASP699 for human V 1B Rs (0.165 nM) was lower than that of TASP0434299 (0.526 nM), whereas its IC 50 values for off-target molecules exceeded 1 μM. PET imaging in monkeys demonstrated that the peak pituitary uptake of 11 C-TASP699 was almost equivalent to that of 11 C-TASP0434299 and that pretreatment with TASP0390325 inhibited the retention of 11 C-TASP699 in a dose-dependent manner, inducing nearly full occupancy at 0.3 mg/kg. Specific radioligand binding was determined as a specific-to-nondisplaceable uptake ratio at equilibrium using radioactivity retentions at 60 min in baseline and blocking studies. This ratio for 11 C-TASP699 was approximately 2.5-fold greater than that of 11 C-TASP0434299. A reversed-phase high-performance liquid chromatography study identified the parent and polar radiometabolites. Affinities of 2 predicted metabolite candidates for V 1B Rs were more than 10 times weaker than that of the parent. Intense autoradiographic labeling of the anterior pituitary with 11 C-TASP699 was inhibited with TASP0390325 in a concentration-dependent manner. Conclusion: 11 C-TASP699 yielded PET images of pituitary V 1B Rs with a higher contrast than 11 C-TASP0434299, supporting the applicability of 11 C-TASP699 in the assessment of neuropsychiatric diseases and dose findings for test drugs in clinical trials., (© 2017 by the Society of Nuclear Medicine and Molecular Imaging.)

In the brain, microglia continuously scan the surrounding extracellular space in order to respond to damage or infection by becoming activated and participating in neuroinflammation. When activated, microglia increase the expression of translocator protein (TSPO) 18 kDa, thereby making the TSPO expression a marker for neuroinflammation. We used the radiotracer [ 11 C]DAA1106 (a ligand for TSPO) and positron emission tomography (PET) to determine the effect of smoking on availability of this marker for neuroinflammation. Forty-five participants (30 smokers and 15 non-smokers) completed the study and had usable data. Participants underwent a dynamic PET scanning session with bolus injection of [ 11 C]DAA1106 (with smokers in the satiated state) and blood draws during PET scanning to determine TSPO affinity genotype and plasma nicotine levels. Whole-brain standardized uptake values (SUVs) were determined, and analysis of variance was performed, with group (smoker vs non-smoker) and genotype as factors, thereby controlling for genotype. Smokers and non-smokers differed in whole-brain SUVs (P=0.006) owing to smokers having 16.8% lower values than non-smokers. The groups did not differ in injected radiotracer dose or body weight, which were used to calculate SUV. An inverse association was found between whole-brain SUV and reported cigarettes per day (P<0.05), but no significant relationship was found for plasma nicotine. Thus, smokers have less [ 11 C]DAA1106 binding globally than non-smokers, indicating less microglial activation. Study findings are consistent with much prior research demonstrating that smokers have impaired inflammatory functioning compared with non-smokers and that constituents of tobacco smoke other than nicotine affect inflammatory processes.

Simple reversible competitive inhibition of nucleotide binding of GTP to Ras family GTPases has long been recognized as an unlikely approach to manipulating the activity of such proteins for experimental or therapeutic purposes. This is due to the high affinity of GTP to GTPases coupled with high cellular GTP concentrations, but also to problems of specificity for the highly conserved binding sites in GTPases. A recent approach suggested that these problems might be overcome by using GDP derivatives that can undergo a covalent reaction with disease specific mutants, in particular addressing inhibition of KRas G12C using GDP equipped with an electrophilic group at the β-phosphate. We show here that a major drawback to this approach is a loss of reversible affinity of such β-modified derivatives for Ras of at least 10 4 compared to GTP and GDP. With the help of a thorough kinetic characterization, we show that this leads to covalent reaction times that are too slow to make the compounds attractive for intracellular use, but that generation of a hypothetical reactive GDP derivative that retains the high reversible affinity of GDP/GTP to Ras might be a viable alternative.

Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through N -dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. Sphingobium baderi DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of S. baderi DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of meaX and meaY are responsible for the initial step of MEA degradation in S. baderi DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the para -position. Transcription of meaX was enhanced remarkably upon induction of MEA or DEA in S. baderi DE-13. Additionally, meaX and meaY were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads. IMPORTANCE Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial degradation is considered an important mechanism in the degradation of these compounds. Bacterial degradation of chloroacetanilide herbicides has been investigated in many recent studies. Pure cultures or consortia able to mineralize these herbicides have been obtained. The catabolic pathway has been proposed, and most key genes involved have been identified. However, the genes responsible for the initiation step (from MEA to hydroxylated MEA or from DEA to hydroxylated DEA) of the downstream pathway have not been reported. The present study demonstrates that a two-component flavin-dependent monooxygenase system, MeaXY, catalyzes the para -hydroxylation of MEA or DEA in sphingomonads. Therefore, this work finds a missing link in the biochemical pathway that carries out the mineralization of chloroacetanilide herbicides in sphingomonads. Additionally, the results expand our understanding of the degradation of a special kind of aniline derivative., (Copyright © 2017 American Society for Microbiology.)

We studied the individual and joint acute toxicity of S-metolachlor (SMOC) and deethylatrazine (DEA - a metabolite of atrazine) on different non-target freshwater crustaceans. We used animals from different ecological groups: two amphipods from surface running water (Gammarus pulex and Gammarus cf. orinos), an isopod from surface stagnant water (Asellus aquaticus) and an amphipod living in groundwater (Niphargus rhenorhodanensis). Organisms were exposed to different levels of SMOC and DEA, alone or in binary mixture. Temperature effect on SMOC toxicity was assessed by exposing G. pulex and N. rhenorhodanensis to SMOC at 11 °C and 15 °C. Studying mortality as the biological endpoint, N. rhenorhodanensis was more resistant than surface water species towards SMOC and DEA. Among surface water species, G. pulex was the most sensitive while Gammarus cf. orinos and A. aquaticus showed similar responses to both compounds. Temperature increase did not change SMOC toxicity but modify the shape and steepness of the dose-response curve. We used a Model Deviation Ratio (MDR) approach to evaluate the predictability of Concentration Addition (CA) and Independent Action (IA) models to mixture toxicity. Results indicated either an additive or an antagonistic or a synergistic interaction depending on the concentrations combination and the test species. Our finding conclusively show the suitability of CA and IA in predicting mixture toxicities but results should be interpreted with caution according to ecological group of exposed species in risk assessment procedures., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Within this study, we designed and synthesized four new oxime compounds of the N-substituted 2-hydroxyiminoacetamide structure and evaluated their interactions with acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Our aim was to explore the possibility of extending the dual-binding mode of interaction between the enzyme and the inhibitor to a so-called triple-binding mode of interaction through the introduction of an additional binding moiety. N-substituted 2-hydroxyiminoacetamide 1 was prepared via BOP catalyzed amidation of hydroxyiminoacetic acid with 3-azido-1-phenylpropylamine. An azide group enabled us to prepare more elaborate structures 2-4 by the copper-catalyzed azide-alkyne cycloaddition. The new compounds 1-4 differed in their presumed AChE peripheral site binding moiety, which ranged from an azide group to functionalized heterocycles. Molecular docking studies revealed that all three binding moieties are involved in the non-covalent interactions with ChEs for all of the four compounds, albeit not always in the complete accordance with the proposed hypothesis. All of the four compounds reversibly inhibited the ChEs with their inhibition potency increasing in the same order for both enzymes (1 < 2 < 4 < 3). A higher preference for binding to BChE (K I from 0.30 μmol/L to 130 μmol/L) over AChE (K I from 50 μmol/L to 1200 μmol/L) was observed for all of the compounds. Compounds were screened for reactivation of cyclosarin-, sarin- and VX-inhibited AChE and BChE., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Background: The neuroinflammatory glial response contributes to the degenerative process in Parkinson's disease (PD). However, the pattern of microglial progression remains unclear., Methods: We evaluated microglial activation in early stage PD patients by quantifying changes in neuroinflammation using PET with [(11)C]DPA713, a selective PET tracer for microglial activation. Eleven PD patients (Hoehn and Yahr stages 1-2) without dementia underwent the [(11)C]DPA713 PET scan two times with 1 year apart. The binding potential (BPND) was estimated with the simplified reference tissue model. Voxelwise and regions of interest analyses were used to compare the regional BPND among groups., Results: Significant increase in [(11)C]DPA713 BPND was found extrastriatally in the occipital, temporal and parietal cortex in PD patients, and the degree of BPND became much higher over the brain regions predominantly in the temporal and occipital cortex 1 year later., Conclusion: The current results indicated that an extrastriatal spreading of microglial activation reflects one of PD pathophysiology occurring at an early stage.