Redesigning the active site of a carboxyl esterase from the archaeon Archaeoglobus fulgidus to improve sensitivity to organophosphorus compounds

Organophosphorus compounds (OPs) are widely used as pesticides because of their ability to inhibit the activity of acetylcholinesterase (AChE) in the nervous system. Thus, AChE is generally used as a biosensor for pesticide detection. Due to the instability of AChE a more stable enzyme would be desirable for robust applications. We investigated the sensitivity of a thermostable carboxylesterase from the archaeon Archaeoglobus fulgidus (AFEST) to seven selected OPs. The IC₅₀ of dichlorvos against AFEST (50.8 ± 2.6 nM) was 10-fold lower than that of the commercially obtained AChE, indicating that AFEST had higher sensitivity. Its sensitivity for the other OPs was lower than AChE. To enhance the sensitivity of AFEST to OPs, site-directed mutations were introduced in the cap domain of AFEST. The sensitivity of mutant N44S/S48V was enhanced toward all seven OPs compared to the wild-type and was higher than AChE for four OPs, including paraoxon (3.3 ± 0.01 nM), dichlorvos (28.0 ± 0.6 nM), profenofos (43.0 ± 1.0 nM), and diazinon (3.0 ± 0.2 nM). The half-lives of AFEST and the mutant N44S/S48V at 37 °C were over 15 d. The interactions between the enzymes and select OPs were investigated by molecular docking. The results demonstrated that AFEST and the mutant N44S/S48V have the potential to be biosensor for OP detection.     

Pyridine sulfonamide as a small key organic molecule for the potential treatment of type-II diabetes mellitus and Alzheimer’s disease: In vitro studies against yeast α-glucosidase,acetylcholinesterase and butyrylcholinesterase

This paper presents the efficient high yield synthesis of novel pyridine 2,4,6-tricarbohydrazide derivatives (4a–4i) along with their α-glucosidase, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition activities. The enzymes inhibition results showed the potential of synthesized compounds in controlling both type-II diabetes mellitus and Alzheimer’s disease. In vitro biological investigations revealed that most of compounds were more active against yeast α-glucosidase than the reference compound acarbose (IC₅₀ 38.25 ± 0.12 μM). Among the tested series the compound 4c bearing 4-flouro benzyl group was noted to be the most active (IC₅₀ 25.6 ± 0.2 μM) against α-glucosidase, and it displayed weak inhibition activities against AChE and BChE. Compound 4a exhibited the most desired results against all three enzymes, as it was significantly active against all the three enzymes; α-glucosidase (IC₅₀ 32.2 ± 0.3 μM), AChE (IC₅₀ 50.2 ± 0.8 μM) and BChE (IC₅₀ 43.8 ± 0.8 μM). Due to the most favorable activity of 4a against the tested enzymes, for molecular modeling studies this compound was selected to investigate its pattern of interaction with α-glucosidase and AChE targets.     

Synthesis,biological evaluation and docking studies of 2,3-dihydroquinazolin-4(1H)-one derivatives as inhibitors of cholinesterases

In search of potent inhibitors of cholinesterases, we have synthesized and evaluate a number of 2,3-dihydroquinazolin-4(1H)-one derivatives. The synthetic approach provided an efficient synthesis of the target molecules with excellent yield. All the tested compounds showed activity against both the enzymes in micromolar range. In many case, the inhibition of both enzymes are higher than or comparable to the standard drug galatamine. With the selectivity index of 2.3 for AChE, compound 5f can be considered as a potential lead compound with a feature of dual AChE/BChE inhibition with IC₅₀ = 1.6 ± 0.10 μM (AChE) and 3.7 ± 0.18 μM (BChE). Binding modes of the synthesized compounds were explored by using GOLD (Genetic Optimization for Ligand Docking) suit v5.4.1. The computed binding modes of these compounds in the active site of AChE and BChE provide an insight into the mechanism of inhibition of these two enzyme.     

Bioaugmentation of carbofuran by Burkholderia cepacia PCL3 in a bioslurry phase sequencing batch reactor

The effectiveness of bioremediation technology in the removal of carbofuran from contaminated soil using a bioslurry phase sequencing batch reactor (SBR) was investigated. A 2-L laboratory glass bottle was used as a bioreactor with a working volume of 1.5 L at room temperature (27 ± 2 °C). One total cycle period of the SBR was comprised of 1 h of fill phase, 82 h of react phase, and 1 h of decant phase. The carbofuran concentration in the soil was 20 mg/kg soil. A carbofuran degrader isolated from carbofuran phytoremediated soil, namely Burkholderia cepacia PCL3 (PCL3) immobilized on corncob, was used as the inoculum. The results revealed that bioaugmentation treatment (addition of PCL3) gave the highest percentage of carbofuran removal (96.97%), followed by bioaugmentation together with biostimulation (addition of molasses) treatment (88.23%), suggesting that bioremediation was an effective technology for removing carbofuran in contaminated soil. Abiotic experiments, i.e. autoclaved soil slurry with corncob and no PCL3 treatment and autoclaved soil slurry with no PCL3 treatment, could adsorb 31.86% and 7.70% of carbofuran, respectively, which implied that soil and corncob could act as sorbents for the removal of carbofuran.     

Novel antioxidant bromophenols with acetylcholinesterase,butyrylcholinesterase and carbonic anhydrase inhibitory actions

In this study, a series of novel bromophenols were synthesized from benzoic acids and methoxylated bromophenols. The synthesized compounds were evaluated by using different bioanalytical antioxidant assays including 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS⁺) radical scavenging assays. Also, reducing power of novel bromophenols were evaluated by Cu²⁺-Cu⁺ reducing, Fe³⁺-Fe²⁺ reducing and [Fe³⁺-(TPTZ)₂]³⁺-[Fe²⁺-(TPTZ)₂]₂+ reducing and ferrous ions (Fe²⁺) chelating abilities. The compounds demonstrate powerful antioxidant activities when compared to standard antioxidant molecules of α-tocopherol, trolox, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT). Also in the last part of this studies novel bromophenols were tested against some metabolic enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE) enzymes and carbonic anhydrase I, and II (hCA I and hCA II) isoenzymes. The newly synthesized bromophenols showed Ki values in a range of 6.78 ± 0.68 to 126.07 ± 35.6 nM against hCA I, 4.32 ± 0.23 to 72.25 ± 12.94 nM against hCA II, 4.60 ± 1.15 to 38.13 ± 5.91 nM against AChE and 7.36 ± 1.31 to 29.38 ± 3.68 nM against BChE.     

Propiconazole induced toxicological alterations in brain of freshwater fish Channa punctata Bloch

Fungicides are a class of pesticides which are used indiscriminately in large amounts and pose a serious threat to the environment. Propiconazole (PCZ) is a systemic foliar fungicide with a broad range of activity. The potential of this fungicide to induce toxicity has not been fully explored. The present study was designed to investigate the dose dependent neurotoxic effect of propiconazole (PCZ), with Channa punctata Bloch as a model organism. Effect of PCZ on the brain specific enzyme activity such as acetylcholinesterase (AChE), monoamine oxidase (MAO) and Na⁺-K⁺-ATPase was determined in the fish brain tissue exposed to sub-lethal concentrations (0.5 and 5 ppm) for 96 h. Also, levels of oxidative stress reflected by various enzymatic and non-enzymatic antioxidants were measured. Neurotransmitter (epinephrine) level was also assessed. PCZ exposure induced oxidative stress as reflected by the significant increase in fish brain lipid peroxidation and protein carbonyl content with decrease in reduced glutathione levels, as well as the significant inhibition of glutathione dependent metabolizing enzymes and CAT activities. In addition, AChE, MAO and Na⁺-K⁺-ATPase activities were significantly lowered along with reduction in epinephrine levels in PCZ exposed fishes than those of the control in a dose dependent manner. Also, histopathological alterations were observed in fish brain of the treated fishes. The results point towards the potential neurotoxicity in the fish caused by PCZ exposure but the application of these findings will need more detailed study before they can be established as special biomarkers for toxicity monitoring the aquatic environment.     

Insecticide resistance monitoring and correlation analysis to select appropriate insecticides against Nilaparvata lugens (Stål), a migratory pest in Korea

Nilaparvata lugens Stål is one of the important migratory pests of rice paddy fields in Korea. Resistance levels to nine insecticides were monitored in 12 local strains and correlation analysis was conducted to determine cross-resistance relationships among the tested insecticides. The local strains revealed 1.3- to 28.0-, 1.6- to 6.0-, 2.8- to 237.0-, 0.6- to 0.9-, and 0.7- to 1.3-fold resistance to carbamates, organophosphates, neonicotinoids, fipronil and etofenprox, respectively. Organophosphate insecticides revealed moderate correlations with benzofuranyl methylcarbamate (r = 0.566–0.614, p > 0.01). Three neonicotinoids were not correlated with each other, but imidacloprid and clothianidin were moderately correlated with several benzofuranyl methylcarbamate and organophosphate insecticides (r = 0.590–0.705, p > 0.05), indicating that unknown common factors (such as detoxification enzymes) might contribute to resistance to both insecticides. Fipronil and etofenprox exhibited low levels of resistance and cross-resistance with other insecticides, suggesting their potential as an effective insecticide for field application. Resistance level monitoring and correlation analysis would be valuable for the selection of appropriate insecticides to control insecticide-resistant N. lugens, a typical migratory pest in Korea.     

Discovery of isoalloxazine derivatives as a new class of potential anti-Alzheimer agents and their synthesis

This article describes discovery of a novel and new class of cholinesterase inhibitors as potential therapeutics for Alzheimer’s disease. A series of novel isoalloxazine derivatives were synthesized and biologically evaluated for their potential inhibitory outcome for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). These compounds exhibited high activity against both the enzymes AChE as well as BuChE. Of the synthesized compounds, the most potent isoalloxazine derivatives (7m and 7q) showed IC₅₀ values of 4.72 μM and 5.22 μM respectively against AChE; and, 6.98 μM and 5.29 μM respectively against BuChE. These two compounds were further evaluated for their anti-aggregatory activity for β-amyloid (Aβ) in presence and absence of AChE by performing Thioflavin-T (ThT) assay and Congo red (CR) binding assay. In order to evaluate cytotoxic profile of these two potential compounds, cell viability assay of SH-SY5Y human neuroblastoma cells was performed. Further, to understand the binding behavior of these two compounds with AChE and BuChE enzymes, docking studies have been reported.     

Selective inhibition of human acetylcholinesterase by xanthine derivatives: In vitro inhibition and molecular modeling investigations

The commonly used beverage and psychostimulant caffeine is known to inhibit human acetylcholinesterase enzyme. This pharmacological activity of caffeine is partly responsible for its cognition enhancing properties. However, the exact mechanisms of its binding to human cholinesterases (acetyl and butyrylcholinesterase; hAChE and hBuChE) are not well known. In this study, we investigated the cholinesterase inhibition by the xanthine derivatives caffeine, pentoxifylline, and propentofylline. Among them, propentofylline was the most potent AChE inhibitor (hAChE IC₅₀ = 6.40 μM). The hAChE inhibitory potency was of the order: caffeine (hAChE IC₅₀ = 7.25 μM) < pentoxifylline (hAChE IC₅₀ = 6.60 μM) ? propentofylline (hAChE IC₅₀ = 6.40 μM). These compounds were less potent relative to the reference agent donepezil (hAChE IC₅₀ = 0.04 μM). Moreover, they all exhibited selective inhibition of hAChE with no inhibition of hBuChE (IC₅₀ > 50 μM) relative to the reference agent donepezil (hBuChE IC₅₀ = 13.60 μM). Molecular modeling investigations indicate that caffeine binds primarily in the catalytic site (Ser203, Glu334 and His447) region of hAChE whereas pentoxifylline and propentofylline are able to bind to both the catalytic site and peripheral anionic site due to their increased bulk/size, thereby exhibiting superior AChE inhibition relative to caffeine. In contrast, their lack of hBuChE inhibition is due to a larger binding site and lack of key aromatic amino acids. In summary, our study has important implications in the development of novel caffeine derivatives as selective AChE inhibitors with potential application as cognitive enhancers and to treat various forms of dementia.     

Effects of exposure to sublethal propiconazole on intestine-related biochemical responses in rainbow trout,Oncorhynchus mykiss

The effect of long-term (30 days) exposure to PCZ (0.2, 50, and 500 μg l^?1) on intestine-related biochemical markers in rainbow trout was investigated. Multiple biomarkers were measured, including digestive enzymes (proteolytic enzymes and amylase), antioxidant responses (TBARS, CP, SOD, CAT, GR and GPx) and energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase). Exposure to 500 μg l^?1 PCZ led to significantly inhibited (p < 0.01) proteolytic enzyme and amylase activity. Activities of the antioxidant enzymes SOD, CAT, and GPx gradually increased at lower PCZ concentrations (0.2 and 50 μg l^?1). At the highest concentration (500 μg l^?1), oxidative stress was apparent as significant higher (p < 0.05) lipid peroxidation and protein carbonyls, associated with an inhibition of antioxidant enzymes activity. Moreover, energy metabolic parameters (RNA/DNA ratio, Na⁺-K⁺-ATPase) were significantly inhibited (p < 0.01) in the intestines of fish exposed to 500 μg l^?1 PCZ, compared with controls. We suggest that long-term exposure to PCZ could result in several responses in intestine-related biochemical markers, which potentially could be used as indicators for monitoring residual PCZ present in the aquatic environment.     

Immobilization of acetylcholinesterase on new modified acrylonitrile copolymer membranes

The three new dual-layer matrices (polyacrylonitrile (PAN) membranes coated with physically bound chitosan (CHI)—PANCHI-A and chemically bound chitosan—PANCHI-B and PANCHI-C) for immobilization of acetylcholinesterase (AChE) were obtained. The chemical-modified PAN membrane (PAN-NaOH + ethylenediamine (EDA)) was used as a base for the prepared dual-layer membranes. For chemical chitosan bound membrane, chitosan was tethered onto the membrane surface to form a dual-layer biomimetic membrane in the presence of glutaraldehyde (GA). The basic characteristics (amount of amino groups, hydrophilicity and transport characteristics) of the chitosan-modified membranes were investigated. The SEM analyses were shown essential morphology change in the different chitosan membranes.The relative activities and V_max of the covalently immobilized enzyme on PANCHI-B and PANCHI-C membranes were higher than that on PANCHI-A membrane and chemical-modified membrane with NaOH + EDA. K_m values for the different modified membranes are lower for the chitosan-treated membranes. The pH and temperature optimum of immobilized enzyme were determined. The bound enzymes on PANCHI-B and PANCHI-C have higher thermal and storage stability in comparison with AChE on PANCHI-A membrane and free enzyme.     

Impact of annealing and heat-moisture treatment on rapidly digestible,slowly digestible and resistant starch levels in native and gelatinized corn,pea and lentil starches

Impact of annealing (ANN) and heat-moisture treatment (HMT) on rapidly digestible starch (RDS), slowly digestible starch (SDS), resistant starch (RS), and expected glycemic index (eGI) of corn, pea, and lentil starches in their native and gelatinized states were determined. ANN was done for 24 h at 70% moisture at temperatures 10 and 15 °C below the onset (T_o) temperature of gelatinization, while HMT was done at 30% moisture at 100 and 120 °C for 2 h. The swelling factor (SF), amylose leaching (AML) and gelatinization parameters of the above starches before and after ANN and HMT were determined. SF and AML decreased on ANN and HMT (HMT > ANN). The gelatinization temperatures increased on ANN and HMT (HMT > ANN). However, the gelatinization temperature range decreased on ANN but increased on HMT. Birefringence remained unchanged on ANN but decreased on HMT. The Fourier transform infrared (FT-IR) absorbance ratio of 1047 cm^?1/1022 cm^?1 increased on ANN but decreased on HMT. ANN and HMT increased RDS, RS and eGI levels and decreased SDS levels in granular starches. HMT had a greater impact than ANN on RDS, RS, and SDS levels. In gelatinized starches, ANN and HMT decreased RDS and eGI, but increased SDS and RS levels. These changes were more pronounced on HMT. This study showed that amylopectin structure and interactions formed during ANN and HMT had a significant impact on RDS, SDS, RS and eGI levels of starches.     

Amperometric biosensor based on a site-specific immobilization of acetylcholinesterase via affinity bonds on a nanostructured polymer membrane with integrated multiwall carbon nanotubes

Acetylcholinesterase (AChE) was immobilized on chemically modified poly-(acrylonitrile-methyl-methacrylate-sodium vinylsulfonate) membranes in accordance with three different methods, the first of which involved random enzyme immobilization via glutaraldehyde, the second one—site-specific enzyme immobilization via glutaraldehyde and Concanavalin A (Con A) and the third method—modified site-specific enzyme immobilization via glutaraldehyde in the presence of a mixture of multiwall carbon nanotubes and albumin (MWCNs + BSA), glutaraldehyde and Con A. Preliminary tests for the activity of immobilized AChE were carried out using these three methods. The third method was selected as the most efficient one for the immobilization of AChE and the prepared enzyme carriers were used for the construction of amperometric biosensors for the detection of acetylthiocholine (ATCh).A five level three factorial central composite design was chosen to determine the optimal conditions for the enzyme immobilization with three critical variables: concentration of enzyme, Concanavalin A and MWCNs. The design illustrated that the optimum values of the factors influencing the amperometric current were C_E: 70 U mL⁻¹; C_{Con A}: 1.5 mg mL⁻¹ and C_MWCN: 11 mg mL⁻¹, with an amperometric current 0.418 μA. The basic amperometric characteristics of the constructed biosensor were investigated. A calibration plot was obtained for a series of ATCh concentrations ranging from 5 to 400 μM. A linear interval was detected along the calibration curve from 5 to 200 μM. The correlation coefficient for this concentration range was 0.995. The biosensor sensitivity was calculated to be 0.065 μA μM⁻¹ cm⁻². The detection limit with regard to ATCh was calculated to be 0.34 μM. The potential application of the biosensor for detection and quantification of organophosphate pesticides was investigated as well. It was tested against sample solutions of Paraoxon. The biosensor detection limit was determined to be 1.39 × 10⁻¹² g L⁻¹ of Paraoxon, as well as the interval (10⁻¹¹ to 10⁻⁸ g L⁻¹) within which the biosensor response was linearly dependant on the Paraoxon concentration. Finally the storage stability of the enzyme carrier was traced for a period of 120 days. After 30-day storage the sensor retained 76% of its initial current response, after 60 days—68% and after 120 days—61%.     

The impact of single and dual hydrothermal modifications on the molecular structure and physicochemical properties of normal corn starch

Effect of single and dual hydrothermal modifications with annealing (ANN) and heat-moisture treatment (HMT) on molecular structure and physicochemical properties of corn starch was investigated. Normal corn starch was modified by ANN at 70% moisture at 50 °C for 24 h and HMT at 30% moisture at 120 °C for 24 h as well as by the combination of ANN and HMT. The apparent amylose content and swelling factor (SF) decreased on ANN and HMT, but amylose leaching (AML) increased. These changes were more pronounced on dual modification. The crystallinity (determined by X-ray diffraction), the gelatinization enthalpy (determined by differential scanning calorimetry) and ratio of 1047 cm^?1/1022 cm^?1 (determined by Fourier transform infrared spectroscopy) slightly increased on ANN and decreased on HMT. The ANN and subsequent HMT (ANN-HMT) resulted in the lowest crystallinity, gelatinization enthalpy and ratio of 1047 cm^?1/1022 cm^?1. The gelatinization temperature range decreased on ANN but increased on HMT. However, the gelatinization range of dually modified starches (ANN-HMT and HMT-ANN) was between ANN starch and HMT starch. Birefringence remained unchanged on ANN but slightly decreased on HMT as well as dual modification. Average chain length and amount of longer branch chains (DP ≥ 37) remained almost unchanged on ANN but decreased on HMT and dual modifications (ANN-HMT and HMT-ANN). HMT and dual modifications resulted in highly reduced pasting viscosity. ANN and HMT as well as dual modifications increased RDS content and decreased SDS and RS content.     

Novel biphenyl bis-sulfonamides as acetyl and butyrylcholinesterase inhibitors: Synthesis,biological evaluation and molecular modeling studies

A series of new biphenyl bis-sulfonamide derivatives 2a–3p were synthesized in good to excellent yield (76–98%). The inhibitory potential of the synthesized compounds on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) was investigated. Most of the screened compounds showed modest in vitro inhibition for both AChE and BChE. Compared to the reference compound eserine (IC₅₀ 0.04 ± 0.0001 μM for AChE) and (IC₅₀ 0.85 ± 0.0001 μM for BChE), the IC₅₀ values of these compounds were ranged from 2.27 ± 0.01 to 123.11 ± 0.04 μM for AChE and 7.74 ± 0.07 to <400 μM for BuChE. Among the tested compounds, 3p was found to be the most potent against AChE (IC₅₀ 2.27 ± 0.01 μM), whereas 3g exhibited the highest inhibition for BChE (IC₅₀ 7.74 ± 0.07 μM). Structure–activity relationship (SAR) of these compounds was developed and elaborated with the help of molecular docking studies.     

Bioaugmentation of carbofuran residues in soil using Burkholderia cepacia PCL3 adsorbed on agricultural residues

Burkholderia cepacia PCL3 (GenBank accession number of EF990634) is a carbofuran degrader isolated from phytoremediated rhizosphere soil in our laboratory. Free and the immobilized PCL3 on corncob and sugarcane bagasse were investigated for their abilities to degrade carbofuran in Basal Salt Medium (BSM) and soil microcosm. The reusability and survival of immobilized PCL3 in comparison to free cells were also examined. Short half-lives (t_1/2) of carbofuran of 3–4 d in BSM were obtained using the isolate PCL3 in both free and immobilized cell forms. Immobilized cells could survive (10⁶–10⁷ cfu ml^?1) through 30 d of incubation, while the number of free cells decreased continuously after 10 d. Immobilized B. cepacia PCL3 could be reused twice without loss in their abilities to degrade carbofuran in BSM, which suggested an advantage of using immobilized cell over free cell. Free and immobilized cells were augmented into soil and showed an effective capability to remediate carbofuran residues, both of which indicated by 5-folds decrease in carbofuran half-lives in augmented soil. Immobilization of PCL3 on corncob and sugarcane bagasse provided the possibilities of reusing the cells as well as improving the cell survival without decreasing carbofuran degradation activity.     

LC–MS quantification of parthenolide and cholinesterase inhibitory potential of selected Tanacetum L. (Emend. Briq.) taxa

The acetonitrile extracts of various Tanacetum L. (Emend. Briq.) taxa from Turkey as well as parthenolide, a sesquiterpene lactone found in Tanacetum species as active substance were investigated for their inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the key enzymes in pathogenesis of Alzheimer's disease, at 100 μg mL⁻¹ using ELISA microplate assay. Most of the extracts displayed a remarkable AChE inhibition where the leaf of Tanacetum argenteum subsp. flabellifolium had the highest inhibition (96.68 ± 0.35%). The extracts had moderate inhibition toward BChE, among which the stem of Tanacetum argyrophyllum var. argyrophyllum-1 exerted the best inhibition (63.81 ± 3.64%). However, parthenolide exhibited low inhibition against both of the enzymes. Total flavonoid content of the extracts was determined spectrophotometrically. Parthenolide, a sesquiterpene lactone, was quantified in these taxa by LC–MS and the leaf of T. argenteum subsp. argenteum possessed the richest parthenolide amount (2.261 ± 0.002%), while most of the species screened were found to contain the required percentage (0.2% minimum) by European Pharmacopeia.     

Effects of three organophosphorus pesticides on population growth and sexual reproduction of rotifer Brachionus calyciflorus Pallas

Effects of organophosphorus pesticides including dichlorvos, triazophos and chlorpyrifos on population growth and sexual reproduction of freshwater rotifer Brachionus calyciflorus were studied by 3-d population growth and 4-d resting egg production tests. The results showed that all the three organophosphorus pesticides influenced significantly the population growth rate, the ratio ovigerous females/non-ovigerous females in the rotifer populations and the resting egg production of the rotifers. Both dichlorvos and chlorpyrifos influenced markedly the mictic rate of the rotifers, but triazophos did not. Compared to the controls, both dichlorvos at 10.0–1000.0 μg/L and chlorpyrifos at 0.01–100.0 μg/L increased the population growth rate, but the reverse was also true for dichlorvos and triazophos both at 10000.0 μg/L. Chlorpyrifos at 10000.0 μg/L made the rotifers dead after 24-hr exposure. Dichlorvos and triazophos both at 10000.0 μg/L, and chlorpyrifos at 1000.0 μg/L all increased the ratio ovigerous females/non-ovigerous females. Both dichlorvos at 10000.0 μg/L and chlorpyrifos at 0.1–100.0 μg/L increased the mictic rate. Dichlorvos at 10.0 μg/L and 100.0 μg/L, and triazophos and chlorpyrifos both at 0.1–100.0 μg/L increased the resting egg production. Both population growth rate and ratio ovigerous females/non-ovigerous females are suitable endpoints for assessing the effects of dichlorvos, triazophos and chlorpyrifos, and mictic rate is a suitable endpoint for monitoring the effects of dichlorvos and chlorpyrifos on the reproduction of the rotifers. Both population growth rate and ratio ovigerous females/non-ovigerous females are more sensitive to dichlorvos and chlorpyrifos than mictic rate.     

Syntheses of coumarin–tacrine hybrids as dual-site acetylcholinesterase inhibitors and their activity against butylcholinesterase,Aβ aggregation,and β-secretase

Exploring small-molecule acetylcholinesterase (AChE) inhibitors to slow the breakdown of acetylcholine (Ach) represents the mainstream direction for Alzheimer’s disease (AD) therapy. As the first acetylcholinesterase inhibitor approved for the clinical treatment of AD, tacrine has been widely used as a pharmacophore to design hybrid compounds in order to combine its potent AChE inhibition with other multi-target profiles. In present study, a series of novel tacrine–coumarin hybrids were designed, synthesized and evaluated as potent dual-site AChE inhibitors. Moreover, compound 1g was identified as the most potent candidate with about 2-fold higher potency (K_i = 16.7 nM) against human AChE and about 2-fold lower potency (K_i = 16.1 nM) against BChE than tacrine (K_i = 35.7 nM for AChE, K_i = 8.7 nM for BChE), respectively. In addition, some of the tacrine–coumarin hybrids showed simultaneous inhibitory effects against both Aβ aggregation and β-secretase. We therefore conclude that tacrine–coumarin hybrid is an interesting multifunctional lead for the AD drug discovery.     

An acetylcholinesterase biosensor based on graphene–gold nanocomposite and calcined layered double hydroxide

In this study, a novel acetylcholinesterase-based biosensor was fabricated. Acetylcholinesterase (AChE) was immobilized onto a glassy carbon electrode (GCE) with the aid of Cu–Mg–Al calcined layered double hydroxide (CLDH). CLDH can provide a bigger effective surface area for AChE loading, which could improve the precision and stability of AChE biosensor. However, the poor electroconductibility of CLDHs could lead to the low sensitivity of AChE biosensor. In order to effectively compensate the disadvantages of CLDHs, graphene–gold nanocomposites were used for improving the electron transfer rate. Thus, the graphene–gold nanocomposite (GN-AuNPs) was firstly modified onto the GCE, and then the prepared CLDH-AChE composite was immobilized onto the modified GCE to construct a sensitive AChE biosensor for pesticides detection. Relevant parameters were studied in detail and optimized, including the pH of the acetylthiocholine chloride (ATCl) solution, the amount of AChE immobilized on the biosensor and the inhibition time governing the analytical performance of the biosensor. The biosensor detected chlorpyrifos at concentrations ranging from 0.05 to 150 μg/L. The detection limit for chlorpyrifos was 0.05 μg/L.