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Objectives: Classical Hirschsprung disease (HD) is defined by the absence of ganglion cells in the rectosigmoid colon. The diagnosis is made from rectal biopsy, which reveals the aganglionosis and the presence of cholinergic hyperinnervation. However, depending on the method of rectal biopsy, the quality of the specimens and the related diagnostic accuracy varies substantially. To facilitate and objectify the diagnosis of HD, we investigated whether software-based identification of cholinergic hyperinnervation in digitalized histopathology slides is suitable for distinguishing healthy individuals from affected individuals., Methods: N = 190 samples of 112 patients who underwent open surgical rectal biopsy at our pediatric surgery center between 2009 and 2019 were included in this study. Acetylcholinesterase (AChE) stained slides of these samples were collected and digitalized via slide scanning and analyzed using two digital imaging software programs (HALO, QuPath). The AChE-positive staining area in the mucosal layers of the intestinal wall was determined. In the next step machine learning was employed to identify patterns of cholinergic hyperinnervation., Results: The area of AChE-positive staining was greater in HD patients compared to healthy individuals (p < 0.0001). Artificial intelligence-based assessment of parasympathetic hyperinnervation identified HD with a high precision (area under the curve [AUC] 0.96). The accuracy of the prediction model increased when nonrectal samples were excluded (AUC 0.993)., Conclusions: Software-assisted machine-learning analysis of AChE staining is suitable to improve the diagnostic accuracy of HD., (© 2024 The Author(s). Journal of Pediatric Gastroenterology and Nutrition published by Wiley Periodicals LLC on behalf of European Society for Pediatric Gastroenterology, Hepatology, and Nutrition and North American Society for Pediatric Gastroenterology, Hepatology, and Nutrition.)

A dual-mode sensor was developed for detecting acetylcholinesterase (AChE) and organophosphorus pesticides (OPs) via bifunctional BSA-CeO 2 nanoclusters (NCs) with oxidase-mimetic activity and fluorescence property. The dual-mode sensor has the characteristics of self-calibration and self-verification, meeting the needs of different detection conditions and provide more accurate results. The colorimetric sensor and fluorescence sensor have been successfully used for detecting AChE with limit of detection (LOD) of 0.081 mU/mL and 0.056 mU/mL, respectively, while the LOD for OPs were 0.9 ng/mL and 0.78 ng/mL, respectively. The recovery of AChE was 93.9-107.2% and of OPs was 95.8-105.0% in actual samples. A novel strategy was developed to monitor pesticide residues and detect AChE level, which will motivate future work to explore the potential applications of multifunctional nanozymes., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Phytochemical study on the rhizomes of Kaempferia parviflora led to the isolation of twenty-three compounds including six phenolic glycosides ( 1 - 6 ), thirteen flavones ( 7 - 19 ), and five phenolic compounds ( 20 - 23 ). Of these, the new compounds were determined to be 2,4-dihydroxy-6-methoxyacetophenone-2 -β- D-apiofuranosyl-(1→6) -β- D-glucopyranoside ( 1 ), 2-hydroxy-4-propionyl-phenyl O-β- D-glucopyranoside ( 2 ), and 4-hydroxy-3,5-dimethoxyacetophenone 8- O-α- L-rhamnopyranosyl-(1→6) -β- D-glucopyranoside ( 3 ) and named as kaempanosides A-C, respectively. Their chemical structures were established based on HR-ESI-MS, 1D and 2D NMR spectra. All compounds 1 - 23 exhibited acetylcholinesterase inhibitory activity with IC 50 values ranging from 57.76 to 253.31 µM.

Although some simple and rapid colorimetric methods have been developed to detect organophosphorus pesticides (OPs), the difficult extraction and easy denaturation of acetylcholinesterase (AChE) are still drawbacks needing to be overcome. Here, we propose a MOF nanozyme-mediated AChE-free colorimetric strategy for the direct detection of OPs. In the presence of OPs (pirimiphos-methyl as a model), the intense blue of oxidized 3,3',5,5'-tetramethylbenzidine (TMB) becomes light due to the quenching effect of OPs towards hydroxyl radicals (˙OH) that are generated by the decomposition of H 2 O 2 catalyzed by the Cu 4 Co 6 ZIF nanozyme with excellent peroxidase (POD)-like activity. The developed colorimetric sensor exhibits assay performance and offers a universal and promising analysis strategy for detecting OPs in practical samples.

Five new biflavonoids, diphybiflavonoids A - E (1-5), were isolated from the roots and rhizomes of Diphylleia sinensis. Their structures were elucidated by extensive spectroscopic data, including UV, IR, HR-ESI-MS and 2D NMR. Their absolute configurations were determined by ECD spectra. All isolated compounds were evaluated for acetylcholinesterase (AChE) inhibitory activity. Compounds 1-4 exhibited the potent AChE inhibitory activities with IC 50 values of 1.62, 2.10, 2.08, and 5.15 μM, respectively. The preliminary structure-activity relationship study indicated that the connection mode (C2-O-C4'''/C3-O-C3''' or C2-O-C3'''/C3-O-C4''') of biflavonoid subunits, and 3-hydroxy group of flavonol subunit were important structural factors for AChE inhibitory activity. Biflavonoids, containing a C2-O-C4'''/C3-O-C3''' or C2-O-C3'''/C3-O-C4''' linkage, can be a potentially useful platform for development of cholinesterase inhibitors., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Anopheles pharoensis has a major role in transmitting several human diseases, especially malaria, in Egypt?. Controlling Anopheles is considered as an effective strategy to eliminate the spread of malaria worldwide. Galaxaura rugosa is a species of red algae found in tropical to subtropical marine environments. The presence of G. rugosa is indicative of the ecosystem's overall health. The current work aims to investigate UPLC/ESI/MS profile of G. rugosa methanol and petroleum ether extracts and its activity against An. pharoensis and non-target organisms, Danio rerio and Daphnia magna., Methods: Galaxaura rugosa specimens have been identified using DNA barcoding for the COI gene and verified as G. rugosa. The UPLC/ESI/MS profiling of G. rugosa collected from Egypt was described. The larvicidal and repellent activities of G. rugosa methanol and petroleum ether extracts against An. pharoensis were evaluated, as well as the toxicity of tested extracts on non-target organisms, Dan. rerio and Dap. magna., Results: The UPLC/ESI/MS analysis of methanol and petroleum ether extracts led to the tentative identification of 57 compounds belonging to different phytochemical classes, including flavonoids, tannins, phenolic acids, phenyl propanoids. Larval mortality was recorded at 93.33% and 90.67% at 80 and 35 ppm of methanol and petroleum ether extracts, respectively, while pupal mortality recorded 44.44 and 22.48% at 35 and 30 ppm, respectively. Larval duration was recorded at 5.31 and 5.64 days by methanol and petroleum ether extracts at 80 and 35 ppm, respectively. A decrease in acetylcholinesterase (AChE) level and a promotion in Glutathione-S-transferase (GST) level of An. pharoensis 3rd instar larvae were recorded by tested extracts. The petroleum ether extract was more effective against An. pharoensis starved females than methanol extract. Also, tested extracts recorded LC 50 of 1988.8, 1365.1, and 11.65, 14.36 µg/mL against Dan. rerio, and Dap. magna, respectively., Conclusions: Using red algae derivatives in An. pharoensis control could reduce costs and environmental impact and be harmless to humans and other non-target organisms., (© 2023. The Author(s).)

Fabrication, characterization and evaluation of the larvicidal potential of novel silk protein (sericin)-based silver nanoparticles (Se-AgNPs) were the prime motives of the designed study. Furthermore, investigation of the sericin as natural reducing or stabilizing agent was another objective behind this study. Se-AgNPs were synthesized using sonication and heat. Fabricated Se-AgNPs were characterized using particle size analyzer, UV spectrophotometry, FTIR and SEM which confirmed the fabrication of the Se-AgNPs. Size of sonication-mediated Se-AgNPs was smaller (7.49 nm) than heat-assisted Se-AgNPs (53.6 nm). Being smallest in size, sonication-assisted Se-AgNPs revealed the significantly highest (F 4,10  = 39.20, p = .00) larvicidal activity against fourth instar lab and field larvae (F 4,10  = 1864, p = .00) of dengue vector (Aedes aegypti) followed by heat-assisted Se-AgNPs and positive control (temephos). Non-significant larvicidal activity was showed by silver (without sericin) which made the temperature stability of silver, debatable. Furthermore, findings of biochemical assays (glutathione-S transferase, esterase, and acetylcholinesterase) showed the levels of resistance in field strain larvae. Aforementioned findings of the study suggests the sonication as the best method for synthesis of Se-AgNPs while the larvicidal activity is inversely proportional to the size of Se-AgNPs, i.e., smallest the size, highest the larvicidal activity. Conclusively, status of the sericin as a natural reducing/stabilizing agent has been endorsed by the findings of this study. RESEARCH HIGHLIGHTS: Incorporation of biocompatible and inexpensive sericin as a capping/reducing agent for synthesis of Se-AgNPs. A novel sonication method was used for the fabrication of Se-AgNPs which were thoroughly characterized by particle size analyzer, UV-visible spectrophotometry, SEM and FTIR. Analysis of enzymatic (GSTs, ESTs) levels in field and lab strains of Aedes aegypti larvae for evaluation of insecticides resistance., (© 2023 Wiley Periodicals LLC.)

Acetylcholinesterase (AChE) plays a crucial role in biological neurotransmission. The aberrant expression of AChE is associated with various neurodegenerative diseases. Therefore, it is of great significance to develop a simple and highly sensitive AChE analysis platform. Herein, a simple colorimetric sensor was constructed for sensitive detection of AChE based on the peroxidase-like catalytic activity of Ni/Co layered double hydroxides (Ni/Co LDHs). In this sensor, the fabricated Ni/Co LDHs possess high peroxidase-like activity, enabling rapid catalysis of o -phenylenediamine (OPD) to produce yellow oxOPD in the presence of H 2 O 2 . This peroxidase-like activity of Ni/Co LDHs was found to be effectively inhibited by the presence of AChE. It is speculated that the combination of AChE on the outer surface of Ni/Co LDHs through non-covalent interaction may cover the active sites and hinder their adsorption to the substrates, leading to the failure of OPD oxidation. As a result, the yellow color from oxOPD is related to the AChE concentration, enabling the direct AChE assay in an equipment-free manner. In addition, the fabricated Ni/Co LDHs could be modified on a paper surface to obtain a paper-based analytical device for visualized colorimetric detection of AChE. The as-proposed sensor shows high sensitivity to AChE with a detection limit down to 6.6 μU mL -1 . Therefore, this naked-eye paper-based sensor is capable of on-site and real-time detection of AChE, and has outstanding application prospects in clinical diagnosis and biomedical fields.

Neurotoxic pesticides are a group of chemicals that pose a severe threat to both human health and the environment. These molecules are also known to accumulate in the food chain and persist in the environment, which can lead to long-term exposure and adverse effects on non-target organisms. The detrimental effects of these pesticides on neurotransmitter levels and function can lead to a range of neurological and behavioral symptoms, which are closely associated with neurodegenerative diseases. Hence, the accurate and reliable detection of these neurotoxic pesticides and associated neurotransmitters is essential for clinical applications, such as diagnosis and treatment. Over the past few decades, acetylcholinesterase (AchE) biosensors have emerged as a sensitive and reliable tool for the electrochemical detection of neurotoxic pesticides and acetylcholine. These biosensors can be tailored to utilize the high specificity and sensitivity of AchE, enabling the detection of these chemicals. Additionally, enzyme immobilization and the incorporation of nanoparticles have further improved the detection capabilities of these biosensors. AchE biosensors have shown tremendous potential in various fields, including environmental monitoring, clinical diagnosis, and pesticide residue analysis. This review summarizes the advancements in AchE biosensors for electrochemical detection of neurotoxic pesticides and acetylcholine over the past two decades., 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 Inc. All rights reserved.)

Background: Hirschsprung's Disease (HD) is characterized by intestinal sub-occlusion and the absence of enteric ganglion cells. A rectal biopsy examination is performed to confirm the diagnosis. In a recent study, we demonstrated that the analysis of 60 sections of rectal mucosa and submucosa stained by H&E may ensure a 90% diagnostic accuracy. Although the need to analyze so many sections makes the process of reading the slides more time-consuming, this encouraged us to study their distribution in the healthy rectal submucosa, to simplify the diagnosis., Objectives: To develop a method that facilitates HD diagnosis by studying the distribution of ganglion cells in the submucosal plexus., Methods: Using the calretinin technique, we studied the distribution of plexuses in 60 fragments of rectal submucosa from 19 cadavers. After the study, the reading method created was used for diagnosis in 47 cases of suspected HD, using H&E staining. The accuracy was verified by comparing the results obtained with H&E to those obtained with the acetylcholinesterase technique, the golden standard in our laboratory., Results: The study of submucosal plexus distribution showed that just by examining the submucosal region every 20 µm, approximately, it is possible to locate a ganglionic plexus, and we have already been able to diagnose HD with 93% accuracy., Conclusion: The study of ganglion cell distribution enabled the creation of a simplified method for reading the slides. The method applied achieved good accuracy and it can be used as an alternative method in HD diagnosis., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2023 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

Objective: To characterize the chemical profile of methanolic crude extract and its fractions (Ethyl acetate, n-butanol and aqueous) using liquid chromatography-mass spectrometry (LC-MS) analysis, to evaluate their biological and pharmacological properties: antioxidant (1, 1-diphenyl-2-pycrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic) (ABTS), galvinoxyle free radical scavenging, reducing power, phenanthroline and β carotene-linoleic acid bleaching assays), enzymes inhibitory ability against several enzymes [acetyl-cholinesterase (AChE), buthyrylcholinesterase (BChE), urease and tyrosinase]., Methods: Secondary metabolites were extracted from Tamarix africana air-dried powdered leaves by maceration, the crude extract was fractionated using different solvents with different polarities (Ethyl acetate, n-butanol and aqueous). The amount of polyphenols, flavonoids and tannins (hydrolysable and condensed) were determined using colorimetric assays. A variety of biochemical tests were carried out to assess antioxidant and oxygen radical scavenging properties using DPPH, ABTS, galvinoxyle free radical scavenging, reducing power, phenanthroline and β carotene-linoleic acid bleaching methods. Neuroprotective effect was examined against acetylcholinesterase and buthy-rylcholinesterase enzymes. The anti-urease and anti-tyrosinase activities were performed against urease and tyrosinase enzymes respectively. The extract's components were identified using LC-MS and compared to reference substances., Results: The results indicated that Tamarix africana extracts presented a powerful antioxidant activity in all assays and exhibited a potent inhibitory effect against AChE and BChE as well as urease and tyrosinase enzymes. LC-MS analysis identified amount of eight phenolic compounds were revealed in this analysis; Apigenin, Diosmin, Quercetin, Quercetine-3-glycoside, Apigenin 7-O glycoside, Rutin, Neohesperidin and Wogonin in methanolic extract and its different fractions of Tamarix africana from leaves., Conclusions: Based on these findings, it is reasonable to assume that Tamarix africana could be considered as a potential candidate for pharmaceutical, cosmetics, and food industries to create innovative health-promoting drugs.

Objective: Alzheimer's disease (AD) is the most prevalent form of dementia among the aging population. Cumulative studies aim to find non-invasive biomarkers in the early stages of AD. Saliva can be obtained easily, and salivary biomarkers have been proven effective in detecting neurodegenerative diseases. To find effective biomarkers in saliva and to help the diagnosis of AD, we performed a meta-analysis focusing on the salivary biomarkers (β-amyloid 1-42 (Aβ 1-42 ), total tau (t-tau), phosphorylated tau (p-tau) and acetylcholinesterase (AChE)) in AD., Methods: We conducted a systematic online search for eligible studies reporting data on salivary biomarkers reflecting Aβ 1-42 , t-tau, p-tau, and AChE in AD cohorts versus controls. Biomarkers' performance was assessed in a random-effects meta-analysis with the ratio of mean (RoM)., Results: A total of thirteen studies were included in the meta-analysis, of them seven involved salivary Aβ 1-42 (271 AD and 489 controls), five involved salivary t-tau (324 AD and 252 controls), four involved salivary p-tau (130 AD and 161 controls), and three involved salivary AChE (81 AD and 54 controls). AD showed significantly higher salivary Aβ 1-42 levels than control (ROM = 1.90 (95% CI 1.28-2.81, P = 0.001), while AD and control did not differ significantly on salivary t-tau, p-tau and AChE (ROM = 0.94, 95% CI 0.67-1.31, P = 0.72; ROM = 0.91, 95% CI 0.56-1.45, P = 0.68; ROM = 0.83, 95% CI 0.24-2.88, P = 0.77; respectively)., Conclusion: The pooled results provide evidence that salivary Aβ 1-42 may serve as a sensitive biomarker for AD; nevertheless, larger AD cohorts are required to further confirm the sensitivity and specificity of salivary Aβ 1-42 for AD diagnosis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

Most organophosphorus pesticide (OP) sensors reported in the literature rely on the inhibition effect of OPs on the activity of acetylcholinesterase (AChE), which suffer from the drawbacks of lack of selective recognition of OPs, high cost, and poor stability. Herein, we proposed a novel chemiluminescence (CL) strategy for the direct detection of glyphosate (an organophosphorus herbicide) with high sensitivity and specificity, which is based on the porous hydroxy zirconium oxide nanozyme (ZrO X -OH) obtained via a facile alkali solution treatment of UIO-66. ZrO X -OH displayed excellent phosphatase-like activity, which could catalyze the dephosphorylation of 3-(2'-spiroadamantyl)-4-methoxy-4-(3'-phosphoryloxyphenyl)-1,2-dioxetane (AMPPD) to generate strong CL. The experimental results showed that the phosphatase-like activity of ZrO X -OH is closely related to the content of hydroxyl groups on their surface. Interestingly, ZrO X -OH with phosphatase-like properties exhibited a unique response to glyphosate because of the consumption of the surface hydroxyl group by the unique carboxyl group of glyphosates and was thus employed to develop a CL sensor for direct and selective detection of glyphosate without using bio-enzymes. The recovery for glyphosate detection of cabbage juice ranged from 96.8 to 103.0%. We believe that the as-proposed CL sensor based on ZrO X -OH with phosphatase-like properties supplies a simpler and more highly selective approach for OP assay and provides a new method for the development of CL sensors for the direct analysis of OPs in real samples.

Introduction: The available literature provides relatively little information on the morphology of the autonomic head ganglia in rodents including their neurochemical codding., Material and Methods: Morphological investigations of the otic ganglion of the chinchilla were performed using the modified acetylcholinesterase method. The cellular structure was investigated with histological techniques and neurochemical properties were studied with the double-labelling immunofluorescence method., Results: Macromorphological investigations allowed the otic ganglion to be identified as a compact, oval agglomeration of neurons and nerve fibers. Multidimensional cross-sections revealed densely arranged neuronal perikarya and two populations of nerve cells differing in size were distinguished. The large cells (40-50 μm) accounted for about 80% of the neurons in the cross-sections. Moreover, a small number of intraganglionic nerve fibers was observed. Immunohistochemical staining revealed that over 85% of the neuronal cell bodies in the otic ganglion contained immunoreactivity to VAChT or ChAT. VIP-immunoreactive perikarya comprised approximately 10% of the ganglionic cells. Double staining revealed the presence of VAChT+ and NOS+ neurons which amounted to about 45% of the nerve cells in the otic ganglion. NOS+ only perikarya comprised approx. 15% of all the neurons. Immunoreactivity to enkephalins, substance P, somatostatin, and galanin was expressed in single nerve cell bodies and nerve fibers except numerous substance P+ intraganglionic nerve fibers. Some of them were stained also for CGRP. Single neurons stained for tyroxine hydroxylase., Conclusions: Our results, compared with findings in other rodent species suggest the existence of interspecies differences in the morphology, cellular structure, and immunohistochemical properties of the head autonomic ganglia in mammals.

The current study reports for the first time the nutritional, fruit volatiles, phytochemical, and biological characteristics of Ferocactus herrerae J. G. Ortega fruits. The nutritional analysis revealed that carbohydrate (20.6%) was the most abundant nutrient followed by dietary fibers (11.8%), lipids (0.9%), and proteins (0.8%). It was rich in vitamins, minerals, essential, and non-essential amino acids. Gas chromatography-mass spectrometry (GC-MS) analysis of the headspace-extracted volatiles showed that 3-methyl octadecane (35.72 ± 2.38%) was the major constituent detected. Spectrophotometric determination of total phenolic and flavonoid contents of the fruit methanolic extract (ME) showed high total phenolic [9.17 ± 0.87 mg/g gallic acid equivalent (GAE)] and flavonoid [4.99 ± 0.23 mg/g quercetin equivalent (QE)] contents. The ME was analyzed using high-performance liquid chromatography with ultraviolet (HPLC-UV), which allowed for both qualitative and quantitative estimation of 16 phenolic compounds. Caffeic acid was the major phenolic acid identified [45.03 ± 0.45 mg/100 g dried powdered fruits (DW)] while quercitrin (52.65 ± 0.31 mg/100 g DW), was the major flavonoid detected. In-vitro assessment of the antioxidant capacities of the ME revealed pronounced activity using three comparative methods; 2, 2-diphenyl-1-picrylhydrazyl (DPPH) (132.06 ± 2.1 μM Trolox equivalent (TE) /g), 2,2'-azino-di(3-ethylbenzthiazoline-6-sulfonic acid (ABTS), (241.1 ± 5.03 uM TE/g), and ferric reducing antioxidant power (FRAP) (258.9 ± 1.75 uM TE/g). Besides, remarkable anti-inflammatory [COX-1 (IC 50  = 20.2 ± 1.1 μg/mL) and COX-2 (IC 50  = 9.8 ± 0.64 μg/mL)] and acetylcholinesterase inhibitory (IC 50  = 1.01 ± 0.39 mg/mL) activities were observed. Finally, our results revealed that these fruits could be used effectively as functional foods and nutraceuticals suggesting an increase in their propagation., (© 2022. The Author(s).)

Organophosphorus nerve agents (OPNAs), such as Sarin (GB), Tabun (GA), Soman (GD) and VX, would cause tremendous harm in military and terrorist attacks, and thus the development of simple methods for the rapid and efficient detection of these hazardous substances is of great necessity. Herein, we present a novel approach for the facile, rapid and sensitive detection of real OPNAs. The detection substrate is fabricated using functionalized silver nanoparticles (AgNPs) immobilized with acetylcholinesterase (AChE) and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). In the absence of OPs, AChE catalyzes the hydrolysis of acetylthiocholine (ATCh) to form thiocholine (TCh), which continues to interact quickly with DTNB to produce a very sensitive Raman probing molecule, TNB. The inhibition of the activity of AChE by OPs could induce an obvious decrease of characteristic Raman peaks of 5-thio-2-nitrobenzoic acid (TNB) at 1335 cm -1 . The introduction of DTNB as an enzyme activity indicator significantly improves the detection sensitivity with distinct characteristic Raman peaks. The LOD of GD, which is one of the most easily aged OPNAs, could reach 0.1 nM due to its strongest inhibition of AChE. Moreover, various OPNAs exhibit different SERS intensities due to their different inhibition capacities of AChE. Hence, the new strategy has great potential in public security early warning and environmental analysis.

Rectal suction biopsy (RSB) is a gold standard for diagnosing Hirschsprung disease (HD). Calretinin staining of RSB is increasingly used by experienced pathologists due to non-complex examination and comparable diagnostic accuracy with acetylcholinesterase (AChE). However, the diagnostic accuracy of calretinin examined by unexperienced pathologists remains to be elucidated. Therefore, we aim to compare diagnostic accuracy of calretinin with AChE on RSB for diagnosing HD when examined by unexperienced pathologists. We prospectively analyzed sections from RSB stained with AChE + HE and calretinin. Blinded examination was done by five unexperienced pathologists (pathology residents) and three experienced pathologists (senior pediatric gastro-enterology pathologists) assessing for the presence of HD. Cases for the study included ones proven to be HD on resection specimens and cases without HD. Diagnostic accuracy was determined calculating area under the curve (AUC), sensitivity, specificity, likelihood ratio, and posttest probability. Fleiss' kappa analysis was performed to assess interobserver agreement between reviewers. Eleven of 18 included patients (61%) were diagnosed with HD. Comparing the diagnostic accuracy of unexperienced pathologists, calretinin versus AChE + HE showed sensitivity of 80.0% versus 74.5% and specificity of 100% versus 65.4%, AUC of 0.87 (0.78-0.96) versus 0.59 (0.45-0.72). Unexperienced pathologists showed substantial agreement with calretinin (kappa 0.72 [0.61-0.84]) and fair agreement with AChE + HE (kappa 0.34 [0.23-0.44]). We found calretinin having higher diagnostic accuracy in diagnosing HD compared to AChE + HE when examined by unexperienced pathologists. Therefore, we recommend to use calretinin as the standard technique for staining RSB in diagnosing HD., (© 2022. The Author(s).)

Analytical method development for the control of pesticide residues occurring in significant dietary foodstuffs is of utmost importance considering their potential impact on consumer health and food market sustainability. Depending on the purpose, either instrumental analysis, mainly chromatographic methods, or screening assays, mostly using biorecognition affinity, are commonly used, featuring different advantages and drawbacks. To practically compare these two different types of analytical strategies, we applied them for the detection of (i) 97 organophosphate (OP) and carbamate (CM) pesticide residues in wheat flour and (ii) carbofuran (a carbamate insecticide) in wheat, rye and maize flour samples. Regarding high-end analysis, an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) method was developed and validated achieving low limits of quantification (LOQs, from 0.002 to 0.040 mg kg -1 ) and a short chromatographic run (12 min). In terms of bioanalytical methods, a fast (17 min) and cost-efficient (∼0.01€ per sample) acetylcholinesterase (AChE) microplate assay for carbofuran screening was utilized. Importantly, carbofuran was the strongest of the 11 OP and CM tested pesticides achieving a half maximal inhibitory concentration (IC 50 ) of 0.021 μM whilst the assay detectability was at the parts per billion level in all three cereal matrices. Based on the attained results, a critical discussion is presented providing the analytical merits and bottlenecks for each case and a wider outlook related to the application of analytical methods in the food safety control analytical scheme.

The supercritical fluid extraction (SFE) of volatile and fixed oil from milled parsley ( Petroselinum crispum L.) seeds, using CO 2 as solvent, is presented in this study. Extraction experiments were carried out in two steps: at pressures of (90 or 300) bar and temperature of 40 °C. The first extraction step, performed at 90 bar, produced a volatile fraction mainly formed by apiole (82.1%) and myristicin (11.4%). The volatile oil yield was 2.6% by weight of the charge. The second extraction step, carried out at 300 bar produced a fixed oil at a yield of 0.4% by weight. The most represented fatty acids in P. crispum fixed oil were 18:1 n-12 (49.9%), 18:2 n-6 (18.2%), 18:1 n-9 (11.8%), and 16:0 (7.4%). In particular, the unsaturated fatty acids 18:1 n-12 and 18:1 n-9 averaged 182.2 mg/g and 92.1 mg/g of oil extract, respectively. The quality of the oils extracted by SFE, in terms of its chemical composition, was compared to the oils obtained by hydrodistillation (HD) in a Clevenger apparatus and by solvent extraction (SE) using n -hexane in a Soxhlet apparatus. The antioxidant properties were determined by means of the ABTS assay. The results indicated that the fixed oil possessed low antioxidant activity (EC 50 = 0.4 mg/mL) and the volatile oil had no antioxidant activity. The total phenolic content, expressed as concentration of gallic acid (gallic acid equivalent, GAE), of the fixed oil was 1.5 mg/g. The fixed oil found to have inhibitory effects against α-glucosidase, the volatile oil is active on acetylcholinesterase (AChE), tyrosinase, and α-glucosidase. Both samples have weak inhibitory activity on α-amylase and no activity on butyrylcholinesterase (BChE).

Aim - based on the clincal experience of monitoring and treatment of teenagers with Hirschsprung's disease (HD), to study clinical aspects of the disease in older children and to establish an adequate diagnostic and treatment policy. The paper is based on the analysis of the results of examination and treatment of 26 children with Hirschsprung's disease, who were first diagnosed with the disease at the age of 10 years and older. There were 19 (73.08%) boys and 7 (26.92%) girls. The following diagnostic methods were used: X-ray examination of the large intestine, ultrasonography of the abdominal organs and the large intestine, rectosigmoidoscopy, morphological examination of biopsy material, immunohistological examination for acetylcholinesterase (AChE). Nonparametric statistical methods were used due to the small sample and the prevalence of the analysis of qualitative rather than quantitative criteria. Hirschsprung's disease in older children has a latent course with long periods of functional compensation. The rectal form of pathology predominates (57.69%). The main diagnostic methods are anamnesis, features of the clinical course of the pathology, irrigoscopy, morphological examination of full-thickness rectal biopsies and immunohistochemical examination of the rectal mucosa for AChE. 84,62% of teenagers underwent one-stage surgery without postoperative complications; 15,38% of patients had indications for colostomy.

Maintaining a long-term continuous and stable reactivator blood concentration to treat organophosphorus nerve agent poisoning using acetylcholinesterase (AChE) reactivator pralidoxime chloride (2-PAM) is very important yet difficult. Because the flexible framework of MIL-88B(Fe) nanoparticles (NPs) can swell in polar solvents, pralidoxime chloride (2-PAM) was loaded in MIL-88B(Fe) NPs (size: ca. 500 nm) by stirring and incubation in deionized water to obtain 2-PAM@MIL-88B(Fe), which had a maximum drug loading capacity of 12.6 wt %. The as-prepared composite was characterized by IR, powder X-ray diffraction (P-XRD), scanning electron microscopy (SEM), ζ-potential, Brunauer-Emmett-Teller (BET), and thermogravimetry/differential thermal analysis (TG/DTA). The results showed that under constant conditions, the maximum drug release rates of 2-PAM@MIL-88B(Fe) in absolute ethanol, phosphate-buffered saline (PBS) solution (pH = 7.4), and PBS solution (pH = 4) at 150 h were 51.7, 80.6, and 67.1%, respectively. This was because the composite showed different swelling behaviors in different solvents. In PBS solution with pH = 2, the 2-PAM@MIL-88B(Fe) framework collapsed after 53 h and released 100% of 2-PAM. For mice after intragastric poisoning with sarin (a neurotoxic agent), an atropine-assisted 2-PAM@MIL-88B(Fe) treatment experiment revealed that 2-PAM@MIL-88B(Fe) continuously released 2-PAM for more than 72 h so that poisoned AChE was continuously and steadily reactivated. The reactivation rate of AChE was 56.7% after 72 h. This composite is expected to provide a prolonged, stable therapeutic drug for the mid- and late-stage treatment of neurotoxic agent poisoning.

Bee pollen (BP) is full of nutrients and phytochemicals, and so it is widely used as a health food and alternative medicine. Its composition and bioactivity mainly depend on the floral pollens. In this work, BP collected by Apis mellifera with different monoculture flowering crops (BP1-6) were used. The types of floral pollen in each BP were initially identified by morphology, and subsequently confirmed using molecular phylogenetic analysis. Data from both approaches were consistent and revealed each BP to be monofloral and derived from the flowers of Camellia sinensis L., Helianthus annuus L., Mimosa diplotricha , Nelumbo nucifera , Xyris complanata , and Ageratum conyzoides for BP1 to BP6, respectively. The crude extracts of all six BPs were prepared by sequential partition with methanol, dichloromethane (DCM), and hexane. The crude extracts were then tested for the in vitro (i) α -amylase inhibitory, (ii) acetylcholinesterase inhibitory (AChEI), and (iii) porcine pancreatic lipase inhibitory (PPLI) activities in terms of the percentage enzyme inhibition and half maximum inhibitory concentration (IC 50 ). The DCM partitioned extract of X . complanata BP (DCMXBP) had the highest active α -amylase inhibitory activity with an IC 50 value of 1,792.48 ± 50.56 µg/mL. The DCM partitioned extracts of C . sinensis L. BP (DCMCBP) and M. diplotricha BP (DCMMBP) had the highest PPLI activities with an IC 50 value of 458.5 ± 13.4 and 500.8 ± 24.8 µg/mL, respectively), while no crude extract showed any marked AChEI activity. Here, the in vitro PPLI activity was focused on. Unlike C . sinensis L. BP, there has been no previous report of M. diplotricha BP having PPLI activity. Hence, DCMMBP was further fractionated by silica gel 60 column chromatography, pooling fractions with the same thin layer chromatography profile. The pooled fraction of DCMMBP2-1 was found to be the most active (IC 50 of 52.6 ± 3.5 µg/mL), while nuclear magnetic resonance analysis revealed the presence of unsaturated free fatty acids. Gas chromatography with flame-ionization detection analysis revealed the major fatty acids included one saturated acid (palmitic acid) and two polyunsaturated acids (linoleic and linolenic acids). In contrast, the pooled fraction of DCMMBP2-2 was inactive but pure, and was identified as naringenin, which has previously been reported to be present in M . pigra L. Thus, it can be concluded that naringenin was compound marker for Mimosa BP. The fatty acids in BP are nutritional and pose potent PPLI activity., Competing Interests: The authors declare there are no competing interests., (©2022 Khongkarat et al.)

Context: Bryophyllum pinnatum (Lam.) Oken (Crassulaceae) is used traditionally to treat many ailments., Objectives: This study characterizes the constituents of B. pinnatum flavonoid-rich fraction (BPFRF) and investigates their antioxidant and anticholinesterase activity using in vitro and in silico approaches., Materials and Methods: Methanol extract of B. pinnatum leaves was partitioned to yield the ethyl acetate fraction. BPFRF was isolated from the ethyl acetate fraction and purified. The constituent flavonoids were structurally characterized using UPLC-PDA-MS 2 . Antioxidant activity (DPPH), Fe 2+ -induced lipid peroxidation (LP) and anticholinesterase activity (Ellman's method) of the BPFRF and standards (ascorbic acid and rivastigmine) across a concentration range of 3.125-100   μg/mL were evaluated in vitro for 4 months. Molecular docking was performed to give insight into the binding potentials of BPFRF constituents against acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)., Results: UPLC-PDA-MS 2 analysis of BPFRF identified carlinoside, quercetin (most dominant), luteolin, isorhamnetin, luteolin-7-glucoside. Carlinoside was first reported in this plant. BPFRF significantly inhibited DPPH radical (IC 50 = 7.382 ± 0.79 µg/mL) and LP (IC 50 = 7.182 ± 0.60 µg/mL) better than quercetin and ascorbic acid. Also, BPFRF exhibited potent inhibition against AChE and BuChE with IC 50 values of 22.283 ± 0.27 µg/mL and 33.437 ± 1.46 µg/mL, respectively compared to quercetin and rivastigmine. Docking studies revealed that luteolin-7-glucoside, carlinoside and quercetin interact effectively with crucial amino acid residues of AChE and BuChE through hydrogen bonds., Discussion and Conclusions: BPFRF possesses an excellent natural source of cholinesterase inhibitor and antioxidant. The material could be further explored for the potential treatment of oxidative damage and cholinergic dysfunction in Alzheimer's disease.

In this work, a pendant droplet-based sensor is developed for the rapid and label-free detection of acetylcholinesterase (AChE) and its inhibitors. The detection limit of AChE reaches 0.17 mU mL -1 . The pIC 50 values of AChE inhibitors such as neostigmine, rivastigmine and galantamine are determined to be 0.45 μM, 0.64 μM and 4.93 μM, respectively.

Eugenia genus is known for its phenolic metabolites, which may influence the progression of the Alzheimer Disease. This study aimed to evaluate the anticholinesterase effects of six Eugenia species from Brazil. Leaves and stems were submitted to maceration (methanol) and partitioned with dichloromethane and ethyl acetate (EtOAc). Samples were screened (200 μg mL -1 ) for the inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). HPLC-ESI-MS/MS analysis allowed the identification of twenty-eight phenolic compounds. Regarding the enzymatic activity, EtOAc fraction of E. mattosii exhibited the best results. Chemical and pharmacological aspects of seasonal E. mattosii extracts were evaluated. The extract from leaves collected in the winter was the most effective for AChE, and the extract from leaves collected in the spring was the most effective for BuChE. Correlating the enzymatic results with the chemical data, it was possible to associate these effects to isoquercitrin, quercetin, catechin, epicatechin, procatecuic acid and myricitrin content.

A convenient, fast, and colorful colorimetric platform with high resolution for acetylcholinesterase (AChE) activity and its inhibitors detection based on the regulation of oxidase-like nanozyme-mediated etching of gold nanorods (AuNRs) has been proposed in this work. MnO 2 nanosheets are selected as the nanozyme. Their excellent oxidase-like activity enables the etching process to proceed smoothly without the usage of unstable H 2 O 2 . When AChE is present, it catalytically hydrolyzes acetylthiocholine (ATCh) to thiocholine (TCh). With high reducing ability, TCh induces the decomposition of MnO 2 nanosheets, causing them to lose their oxidase-like activity. Thus, the etching of AuNRs is hampered. Consequently, with the increasing concentration of AChE, an apparent change in the AuNRs solution color is observed. The proposed platform achieves high-sensitivity detection of AChE (limit of detection = 0.18 mU/mL). Furthermore, the proposed platform also has been demonstrated its applicability for its inhibitors detection. Benefiting from the advantages of convenient and high resolution of visual readout, the proposed platform holds great potential for the detection of AChE and its inhibitors in clinical diagnosis.

Background: Increased acetylcholinesterase (AChE) activity on frozen sections of rectal mucosal biopsies accurately diagnoses Hirschsprung disease (HD). But the quest for a biomarker in blood as a screening test prompts one to look for AChE in blood and study its role in HD diagnosis., Aim: To develop a low-cost reliable method to estimate the AChE activity in plasma and red blood cells (RBCs) in normal children (control) and study its role in HD (test)., Materials and Methods: Optimized method derived after modifying and standardizing known AChE assay protocols for blood were employed on 30 controls to define the AChE cut-off range, on 40 suspected HD cases to categorize them as HD/non-HD based on cut-off values and later compared with gold standard tissue AChE histochemistry of rectal mucosal biopsies., Results: An optimal in-house modified methods of Ellman's was found best suited to analyze plasma AChE activity, method by Wilson and Henderson was optimal for extraction and AChE estimation in RBCs. AChE levels (controls) obtained were 1.03 ± 0.31 U/mL and 5.17 ± 1.52 U/mL in plasma and RBCs, respectively while the plasma AChE was 1.35 ± 0.84 U/mL (HD) and 1.62 ± 0.85 U/mL (non-HD) while RBC AChE was 4.29 ± 3.2 U/mL (HD) and 6.48 ± 4.31 U/mL (non-HD). Sensitivity was 66.67% and 55.56%, specificity was 22.73% and 45.45%, and an accuracy rate of 42.5% and 50% for plasma and RBC, respectively., Conclusions: Mutually exclusive AChE activity range identified for test blood samples overlapped with the normal and hence, not considered a diagnostic tool for HD., Competing Interests: None

The cetacean hippocampal formation has been noted to be one of the smallest relative to brain size of all mammals studied. This region, comprised of the dentate gyrus, hippocampus proper, subiculum, presubiculum, parasubiculum and the entorhinal cortex, is important in learning, memory, and navigation. There have been a number of studies detailing the distribution of acetylcholinesterase (AChE) in the hippocampal formation of terrestrial mammals with the goal of gaining a greater understanding of some aspects of the cholinergic innervation to this region, as well as its parcellation. The present study was undertaken to describe the organization, cytoarchitecture, and distribution of AChE in the hippocampal formation of the Atlantic white-sided dolphin (AWSD) with the view to understand similarities and differences between this aquatic mammal and terrestrial mammals. Nissl-staining demonstrated cytoarchitecture of the hippocampal formation in the AWSD comparable to that reported in other cetaceans. In addition, the AWSD had a rich pattern of AChE staining that distinctly varied between regions and laminae. A number of differences in the distribution of AChE staining in areas comparable to those of terrestrial species reported suggested possible alterations in connectivity of this region. Overall, however, AChE-staining suggested that cholinergic innervation, neural pathways and function of the hippocampal formation of the AWSD is conserved, similar to other mammals., (© 2020 Wiley Periodicals LLC.)

Dietary restriction (DR), as a natural intervention, not only benefits the neuroendocrine system, but also has an antiaging action. Acetylcholinesterase (AChE) is one of the most important bioactive substances and plays a major part in choline changes in the aging process. Thus, we aim to evaluate the effect of DR on AChE in the brains of aging animals. In this study, we synthesize a NIR fluorescent probe BD-AChE for the real-time and in situ monitoring of AChE level changes in living cells and living mice, notably in brains. In situ visualization with BD-AChE verified a decrease in the AchE level in the brains of mice aging models. Evidently, the prepared probe has the excellent capability of measuring AChE variation in the brains of aging mice with DR via NIR fluorescence bioimaging, indicating that long-term DR can effectively affect AChE levels in the brain. The attenuation of AChE level in the brain of aging mice after DR could be helpful in infering the advantageous impact of DR on age-related neurodegenerative disease, as a better treatment alternative in the future.

Hirschsprung's disease (HD) is a congenital disease manifesting various degrees of functional bowel obstruction caused by the absence of enteric ganglion cells, which are usually absent in the colonic segment of the HD patient. Because the aganglionic segment of HD always includes the rectum, pathological diagnosis can be made using a rectal sample. HD should be diagnosed as early as possible because serious complications, such as acute enterocolitis or toxic megacolon, can develop without a definitive diagnosis and appropriate treatment. In the mid-1900s, HD was diagnosed by HE staining of specimens obtained by full-thickness biopsy. Since then, the combination of rectal mucosal biopsy and rubeanic acid-amplificated AChE staining has been brought about by the following milestones: the discovery that the submucosal plexus and the intermuscular plexus had the same level of nerve migration; the findings of research on acetylcholine (ACh) and acetylcholinesterase (AChE) in the intestinal tract; and the establishment of a rubeanic acid amplification method. Consequently, the diagnostic rate of HD improved dramatically in the 1980s. This review outlines the history of diagnostic methods for HD, the roles of ACh and AChE in the intestine, and the method of AChE staining.

Ratiometric fluorescent sensors are powerful tools for quantitative analyses. However, gold nano-clusters (AuNCs) as typical fluorophores in ratiometric sensors have some disadvantages, such as low luminous efficiency. In this study, a highly sensitive ratiometric fluorescence sensor was fabricated by the combination of AuNCs and fluorescein (FL), and the photonic crystals (PhCs) were used to selectively enhance the fluorescence intensity of AuNCs. This fluorescence sensor was used for the sensitive detection of acetylcholinesterase (AChE) and its inhibitor paraoxon. AChE can catalyze the hydrolysis of acetylthiocholine (ATCh) to form thiocholine (TCh), which can induce the fluorescence quenching of AuNCs while having no obvious influence on the fluorescence intensity of FL. AChE can be determined in the range from 0.1 to 25 mU mL-1 with a limit of detection (LOD) of 0.027 mU mL-1, and paraoxon can be determined in the range of 0.06 to 60 ng mL-1 with a LOD 0.025 ng mL-1. This method, as a new way to selectively improve the fluorescence signal of one of the fluorophores in the ratiometric sensor, would be a promising strategy for the sensitive determination of AChE and its inhibitor.

A reversible fluorescence probe for acetylcholinesterase activity detection was developed based on water soluble perylene derivative, N,N'-di(2-aspartic acid)-perylene-3,4,9,10-tetracarboxylic diimide (PASP). Based on the photo-induced electron transfer (PET), PASP fluorescence in aqueous is quenched after combining with copper ions (Cu 2+ ). Acetylcholinesterase (AChE) is well known to catalyze the hydrolysis of acetylcholine (ATCh) to produce thiocholine, whose affinity is strong enough to capture Cu 2+ by thiol (-SH) group from the complex PASP-Cu, resulting in the fluorescence signal of PASP recovers up to 90%. This optical switch is highly sensitive depended on the coordination and dissociation between PASP and Cu 2+ . We proposed its application for AChE activity detection, as well as its inhibitor screening. According to the change of fluorescence intensity, quantifying the detection limit of AChE was 1.78 mU·mL -1 . Classical inhibitors, tacrine and organophosphate pesticide diazinon, were further evaluated for drug screening. The IC 50 value of tacrine was calculated to be 0.43 μM, and the detection limit of diazinon was 0.22 μM. Both of these performances were much better than previous results, revealing our probe is sensitive and reversible for screening applications., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The excessive expression of cholinesterases (ChEs) directly disturbs the metabolism of acetylcholine (ACh), causing disordering neurotransmission in the brain or even Alzheimer's disease and cancer. However, the variation of ChEs including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in brain glioma has not yet been investigated. Therefore, the development of a suitable method for in situ imaging ChEs in brain tissues to understand the physiological functions of ChEs in depth is very important. Herein, a new near-infrared emission fluorescent probe ( IPAN ) for visualization of ChE activity was developed. IPAN exhibits ultrafast response to ChEs, low detection limit for AChE (0.127 U/mL) and BChE (0.0117 U/mL), and a large Stokes shift with near-infrared emission. Based on these excellent attributes, the IPAN was effectively utilized for imaging the fluctuations of ChE activity in the apoptosis cells and zebrafish. Notably, by utilizing the unique probe IPAN , we observed a significant enhancement of ChE activity in the tumor cells and brain glioma, for the first time. We believe that this interesting finding could provide a powerful guidance for tumor resection in the future.

In this study, by rationally designing the stimulus response of graphene quantum dot (GQD)-sensitized terbium/guanine monophosphate (Tb/GMP) infinite coordination polymer (ICP) nanoparticles, we have constructed a smartphone-based colorimetric assay with ratiometric fluorescence, which could be applied for the detection of acetylcholinesterase (AChE) and organophosphorus pesticides (OPs) directly. First, GQDs with abundant functional groups were chosen as the guest, which not only could be used as one of the signal readouts but also served as the antenna ligand to further sensitize the fluorescence of the host Tb/GMP. Upon being excited at 330 nm, the green fluorescence of the Tb/GMP host is highly enhanced, while the blue fluorescence of GQDs is suppressed due to the confinement of the ICP host. With the presence of thiocholine (TCh), an enzymatic product hydrolyzed from acetylthiocholine (ATCh) by AChE, the competitive coordination of Tb 3+ between GMP and TCh results in the collapse of the ICP network and thereby the release of GQDs into the solution; thus, the fluorescence of Tb/GMP turns off and the fluorescence of GQDs turns on. The dual-responsive ratiometric fluorescent intensity change leads to the corresponding green-to-blue fluorescent color change obviously, which constitutes a novel mechanism for the colorimetric analysis of AChE. Moreover, when OPs are subsequently introduced, the activity of AChE is blocked, thus preventing the stimulus response of GQD@Tb/GMP ICP nanoparticles, leading to the fluorescent color change from greenish-blue to green, which could also be employed for OP detection. Benefitting from the high sensitivity, good reliability, and the obvious color changes, the method demonstrated here is a promising candidate to realize smartphone-based point-of-use applications, which is of great importance for timely clinical diagnosis and treatment of OPs related to health issues with AChE as an exposure biomarker in less industrialized countries, in remote settings, or even in home care services.

The aim of this study was to investigate the combination effect of exercise training and eugenol supplementation on the hippocampus apoptosis induced by CPF. 64 adult male albino rats were randomly selected and devided into eight groups of eight including: control, exercise (EXE), chlorpyrifos (CPF), Control + Oil (Co + Oil), Control + DMSO (Co + DMSO), chlorpyrifos + eugenol (CPF + Sup), chlorpyrifos + exercise (CPF + Exe) and, chlorpyrifos + exercise + eugenol (CPF + Exe + Eu). Four experimental groups received intraperitoneal injection (5 days a week) of 3.0 mg/kg body weight CPF in DMSO for 6 consecutive weeks. The exercise groups performed aerobic 5 days per week over 4 weeks. Eugenol were administered by gavage. Finally, the animals were sacrificed using CO 2 gas (a half of the rats were anesthetized with ketamine and xylazine and then perfused) to evaluate hippocampus histology and parameters. The results of this study showed that CPF injection significantly decreased BDNF, AChE and ATP in CA1 area of the hippocampus (p ˂ 0.05). Also, CA1 apoptosis by tunnel assay, it was found that CPF receiving groups with different dosage, showed a significant increase compared to other groups, which was confirmed by increasing cytochrome C and procaspase-3 in CPF groups (p ˂ 0.05). The result of this study show that 4 weeks of exercise training and eugenol supplementation does not improve the destructive effects of CPF in CA1 area of the hippocampus. As a result, it is recommended that future studies longer periods for treatment with exercise and eugenol supplementation.

Acetylcholine (ACh)-mediated vagal transmission as well as nonneuronal ACh release are considered cardioprotective in pathological situations with increased sympathetic drive such as ischemia-reperfusion and cardiac remodeling. ACh action is terminated by hydrolysis by the cholinesterases (ChEs), acetylcholinesterase, and butyrylcholinesterase. Both ChEs exist in multiple molecular variants either soluble or anchored by specific anchoring proteins like collagen Q (ColQ) anchoring protein and proline-rich membrane anchoring protein (PRiMA). Here we assessed the expression of specific ChE molecular forms in different heart compartments using RT-qPCR. We show that both ChEs are expressed in all heart compartments but display different expression patterns. The acetylcholinesterase-T variant together with PRiMA and ColQ is predominantly expressed in rat atria. Butylcholinesterase is found in all heart compartments and is accompanied by both PRiMA and ColQ anchors. Its expression in the ventricular system suggests involvement in the nonneuronal cholinergic system. Additionally, two PRiMA variants are detected throughout the rat heart.

Objective: The feasibility of the use of WHO impregnated paper and biochemical assays to determine lethal concentrations (LC50 and LC99) and insecticide metabolic enzyme levels of Triatoma dimidiata., Materials and Methods: LC50 and LC99 were calculated with WHO papers impregnated at different concentrations of malathion, propoxur and deltamethrin; the percentage of insensitive acetylcholinesterase (iAChE); and the levels of esterases, glutathione S-transferases, and monooxygenases in laboratory nymphs of the first stage (5 to 7 days), were undertaken using the WHO biochemical assays., Results: Respectively the LC50 and LC99 μg/cm2 obtained for malathion were 43.83 and 114.38, propoxur 4.71 and 19.29, and deltamethrin 5.80 and 40.46. A 30% of the population had an iAChE, and only a few individuals had high P450 and β-eterase levels., Conclusions: Impregnated papers and biochemical tests developed by WHO for other insects, proved to be feasible methods in monitoring insecticide resistance and metabolic enzymes involved in T. dimidiata., Competing Interests: Declaration of conflict of interests. The authors declare that they have no conflict of interests.

Objective: To determine the insecticide resistance status of Ae. aegypti and Ae. albopictus from Tapachula, México., Materials and Methods: Mosquito eggs were collected with the use of ovitraps and CDC susceptibility bioassays and biochemical assays were conducted to determine resistance levels and resistance mechanisms, respectively., Results: Ae. aegypti showed resistance to deltamethrin and permethrin (PYRs), malathion, chlorpyrifos and temephos (OP), and to bendiocarb (CARB), while Ae. albopictus showed resistance to malathion and to a lesser intensity to chlorypirifos, temephos, permethrin and deltamethrin. Both species showed high levels of P450 and GSTs, while levels of esterases varied by species and collection site. Altered acethilcholinesterase was detected in both species., Conclusions: In an urban habitat from Tapachula, Chiapas, Mexico where vector control using insecticides takes place, Ae. aegypti and Ae. albopictus are only susceptible to propoxur., Competing Interests: Declaration of conflict of interests. The authors declare that they have no conflict of interests.

Our goal was to investigate the neuroprotective effects of galantamine in a mouse model of blast-induced indirect traumatic optic neuropathy (bITON). Galantamine is an FDA-approved acetylcholinesterase inhibitor used to treat mild-moderate Alzheimer's disease. We exposed one eye of an anesthetized mouse to repeat bursts of over-pressurized air to induce traumatic optic neuropathy. Mice were given regular or galantamine-containing water (120 mg/L) ad libitum, beginning immediately after blast and continuing for one month. Electroretinograms and visual evoked potentials were performed just prior to endpoint collection. Histological and biochemical assessments were performed to assess activation of sterile inflammation, axon degeneration, and synaptic changes. Galantamine treatment mitigated visual function deficits induced by our bITON model via preservation of the b-wave of the electroretinogram and the N1 of the visual evoked potential. We also observed a reduction in axon degeneration in the optic nerve as well as decreased rod bipolar cell dendritic retraction. Galantamine also showed anti-inflammatory and antioxidant effects. Galantamine may be a promising treatment for blast-induced indirect traumatic optic neuropathy as well as other optic neuropathies., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Nanozymes are one of the ideal alternatives to natural enzymes for various applications. The rational design of nanozymes with improved catalytic activity stimulates increasing attention to address the low activity of current nanozymes. Here, we reported a general strategy to fabricate the Co-based homobimetallic hollow nanocages (HNCs) (C-CoM-HNC, M = Ni, Mn, Cu, and Zn) by ion-assistant solvothermal reaction and subsequent low-temperature calcination from metal-organic frameworks. The C-CoM-HNCs are featured with HNCs composed of interlaced nanosheets with homogeneous bimetallic oxide dispersion. The hierarchical structure and secondary metallic doping endow the C-CoM-HNC highly active sites. In particular, the Cu-doped C-CoCu-HNCs nanostructures exhibit superior performances over the other C-CoM-HNC as both the oxidase mimicking and peroxymonosulfate (PMS) activator. A sensitive bioassay for acetylcholinesterase (AChE) was established based on the excellent oxidase-like activity of C-CoCu-HNC, offering a linear detection range from 0.0001 to 1 mU/mL with an ultralow detection limit of 0.1 mU/L. As the PMS activator, the C-CoCu-HNC was applied for targeted organic pollutant (rhodamine B, RhB) degradation. A highly efficient RhB degradation was realized, along with good adaptability in a wide pH range and good reusability during the eight-cycle run. The results suggest that C-CoCu-HNC holds a practical potential for clinical diagnostics and pollution removal. Further density functional theory calculation reveals that Cu doping leads to a tighter connection and more negative adsorption energy for O 2 /PMS, as well as an upshifted d -band center in the C-CoCu-HNCs nanostructures. These changes facilitated the adsorption of O 2 /PMS on the C-CoCu-HNC surface for dissociation. This work not only offers a promising multifunctional nanozyme catalyst for clinical diagnostics and pollution removal but also gives some clues for the further development of novel nanozymes with high catalytic activities.

We propose a new capillary electrophoresis (CE)-based open-tubular immobilized enzyme microreactor (OT-IMER) and its application in acetylcholinesterase (AChE) assays. The IMER is fabricated at the capillary inlet (reactor length of ∼1 cm) with the inner surface modified by a micropore-structured layer (thickness of ∼220 nm, pore size of ∼15-20 nm). The use of IMER accomplishes the enzymatic reaction and separation/detection of the products in the same capillary within 3 min. The feasibility of the proposed method is evaluated via online analysis of the activity and inhibition of AChE enzymes. Such method exhibits good reproducibility with relative standard deviation (RSD) of less than 4% for 20 runs, and the enzyme remains over 82% of the initial activity after usage of 7 days. The IMERs are successfully applied to detect the organophosphorus pesticide, paraoxon, in three types of vegetable juice samples with a limit of detection of as low as 61 ng mL -1 . Results show that the spiked samples are in the range of 89.6-105.9% with RSD less than 2.7%, thereby indicating its satisfactory level of accurate and reliable analysis of real samples by using the proposed method. Our study indicates that, with combination of advantages of both porous-layer capillary and CE OT-IMER, the proposed method is capable to enhance enzymatic reactions and to achieve rapid analysis with simple instrumentation and operation, thus would pave the way for extensive application of CE-based IMERs in a variety of bioanalysis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Plants are an important source of natural iridoids. This study demonstrates for the first time the acetylcholinesterase (AChE) inhibitory activity of iridoids belonging to the class of antirrhinosides. As iridoids distinguish the chemical composition of most species of the Plantaginaceae family, the active AChE inhibitors were investigated in the hydro-alcoholic extract of Anarrhinum pubescens Fresen. High-performance thin-layer chromatography (HPTLC) in combination with the AChE inhibition assay is a time and material saving methodology, and thus was employed to directly point to the individual enzyme inhibitors occurring in the plant. The effect-directed screening successfully discovered three active metabolites. These were characterized as antirrhinoside-derived iridoids. Two of these are here reported as newly isolated natural compounds. Identification of the two new metabolites was based on analysis of their collected spectroscopic data (HRMS, 1D and 2D NMR). Their structures were elucidated to be 6-O-, 6'-O-di-trans-cinnamoyl-antirrhinoside (1) and 5-O-, 6-O-difoliamenthoyl-antirrhinoside (3), while the previously known compound 6-O-foliamenthoyl-(6'-O-cinnamoyl)-antirrhinoside (2) was assigned by extensive analysis of its HRMS and HRMS/MS data. The activity of the isolated compounds was referred to the known AChE inhibitor rivastigmine, i.e. their activity were calculated and expressed as values equivalently to rivastigmine. This neuroprotective potential of iridoids mediated through AChE inhibition promote them to compete as natural curatives for neurodegenerative disorders like Alzheimer's disease., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The concept that catalytic enzymes can act as molecular machines transducing chemical activity into motion has conceptual and experimental support, but experimental support has involved oligomeric enzymes, often studied under conditions where the substrate concentration is higher than biologically relevant and accordingly exceeds k M , the Michaelis constant. Urease, a hexamer of subunits, has been considered to be the gold standard demonstrating enhanced diffusion. Here we show that urease and certain other oligomeric enzymes dissociate above k M into their subunits that diffuse more rapidly, thus providing a simple physical mechanism that contributes to enhanced diffusion in this regime of concentrations. Mindful that this conclusion may be controversial, our findings are supported by four independent analytical techniques: static light scattering, dynamic light scattering (DLS), size-exclusion chromatography (SEC), and fluorescence correlation spectroscopy (FCS). Data for urease are emphasized and the conclusion is validated for hexokinase, acetylcholinesterase, and aldolase. For hexokinase and aldolase no enhanced diffusion is observed except under conditions when these oligomeric enzymes dissociate. At substrate concentration regimes below k M at which acetylcholinesterase and urease do not dissociate, our finding showing up to 10% enhancement of the diffusion coefficient is consistent with various theoretical scenarios in the literature.

Single-atom catalysts (SACs) have attracted extensive attention in the catalysis field because of their remarkable catalytic activity, gratifying stability, excellent selectivity, and 100% atom utilization. With atomically dispersed metal active sites, Fe-N-C SACs can mimic oxidase by activating O 2 into reactive oxygen species, O 2 - • radicals. Taking advantages of this property, single-atom nanozymes (SAzymes) can become a great impetus to develop novel biosensors. Herein, the performance of Fe-N-C SACs as oxidase-like nanozymes is explored. Besides, the Fe-N-C SAzymes are applied in biosensor areas to evaluate the activity of acetylcholinesterase based on the inhibition toward nanozyme activity by thiols. Moreover, this SAzymes-based biosensor is further used for monitoring the amounts of organophosphorus compounds., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This work describes a new simultaneous on-flow dual parallel enzyme assay based on immobilized enzyme reactors (ICERs) with mass spectrometry detection. The novelty of this work relies on the fact that two different enzymes can be screened at the same time with only one single sample injection and in less than 6 min. The system consisted of two immobilized capillary enzyme reactors (ICERs). More specifically, the ICERs comprised two different enzymes that were accommodated in parallel and were placed between a liquid chromatography (LC) system and a mass spectrometer (MS). The resulting system could be adapted to other types of enzyme reactors with different supports. All the elements in the system were interfaced by means of two 10-port/two-position switching valves. Different tubing dimensions allowed us to monitor the activity of each enzyme independently during the same analysis. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) bioreactors were chosen as proof of concept. Acetylcholine (ACh) was used as substrate; the area of its protonated enzymatic hydrolysis product ion, choline, [M+H] + m/z 104.0, was monitored in the presence and absence of the standard cholinesterase inhibitor galantamine. This method proved to be an interesting tool for fast, simultaneous, and independent label-free dual enzyme inhibitor assay., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Acetylcholinesterase (AChE) plays a vital role in nerve conduction through rapidly hydrolyzing the neurotransmitter acetylcholine (ACh) and is correlated with Alzheimer's disease. In this work, a label-free single-particle enumeration (SPE) method for the quantitative detection of acetylcholinesterase (AChE) activity is developed. The design is based on the fluorescence resonance energy transfer (FRET) between fluorescent conjugated polymer nanoparticles (FCPNPs) and MnO2 nanosheets. The fluorescence of FCPNPs can be effectively quenched by MnO2 nanosheets via hydrogen bonding interaction. In the presence of acetylcholinesterase (AChE), acetylthiocholine (ATCh) could be hydrolyzed to thiocholine (TCh), which can reduce MnO2 to Mn2+ and trigger the decomposition of MnO2 nanosheets. As a result, the fluorescence of FCPNPs is restored. Taking advantage of the superior brightness and stable fluorescence emission from individual FCPNPs, the accurate quantification of AChE is achieved by statistically counting the fluorescent particles on the glass slide surface. A linear range from 5 to 1600 μU mL-1 is obtained for AChE assay and the limit-of-detection (LOD) is 1.02 μU mL-1, which is far below the spectroscopic measurements in bulk solution. In the human serum sample, satisfactory recovery efficiencies are determined in a range of 91.0%-103.0%. Furthermore, pesticide carbaryl as an inhibitor of AChE activity was detected. The LOD is 1.12 pg mL-1 with linear responses ranging from 5 to 300 pg mL-1, which demonstrates the feasibility of this approach for AChE inhibitor screening. As a consequence, the label-free SPE-based method affords a promising platform for the sensitive detection of target molecules in the future.

This paper presents a new probe for fluorescence detection of the acetylcholinesterase (AChE) activity based on molecularly imprinted polymer (MIP) coated carbon dots (C-dots) composite. The C-dots were hydrothermally synthesized with grafted silica surface and sealed with molecularly imprinted polymers in silica pores (MIP@C-dots) in situ. Removed the original template molecules, the MIP@C-dots composite exhibits quite high selectivity for acetylthiocholine (ACh). With AChE, its substrate ACh will be hydrolyzed into thiocholine and the fluorescence signals exhibit a dramatic decrease at 465 nm, Under optimal conditions, the fluorescent probe shows sensitive responses to AChE in the range of 0.01-0.6 mU/mL. The detection limits of AChE are as low as 3 µU/mL. These experiments results validate the novel fluorescent probe based on MIP@C-dots composite, paving a new way to evaluation of AChE activity and Screening inhibitors.

Aims: Protective efficacy of N‑acetylcysteine (NAC) was assessed against sub-acute diisopropyl phosphorofluoridate (DFP) poisoning in mice., Main Methods: Mice were allocated into nine groups of six each: vehicle control; DFP (0.125 LD 50  ≈ 0.483 mg/kg bwt, s.c.); DFP + Atropine (ATR, 10 mg/kg bwt, i.p., 0 min); DFP + Pralidoxime (2-PAM, 30 mg/kg bwt, i.m., 0 min); DFP + NAC (150 mg/kg bwt, i.p., -60 min); DFP + ATR + NAC; DFP + 2-PAM + NAC; DFP + ATR + 2-PAM; and DFP + ATR + 2-PAM + NAC. Animals received various treatments for 21 d daily. Plasma butyrylcholinesterase (BChE) was measured after 7, 14 and 21 d of exposure. Brain acetylcholinesterase (AChE) and reduced glutathione (GSH), malondialdehyde (MDA), glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD) were measured (brain, liver and kidney) after 21 d of exposure. Histopathology, immunohistochemistry, and Western blot for inducible nitric oxide synthase (iNOS) and c-fos were also performed., Key Findings: DFP significantly reduced BChE and AChE levels. Diminished GSH, CAT, SOD (brain and liver), GPx, GR, and elevated MDA (Brain) levels were also observed. DFP caused notable histopathology (brain, liver and kidney) and over expression of iNOS, and c-fos proteins (brain). NAC enhanced the protective efficacy of ATR and 2-PAM in most parameters, without any appreciable protection in iNOS and c-fos expression., Significance: NAC as an adjunct with ATR and 2-PAM, exhibited marked beneficial effects against sub-acute DFP poisoning, indicating its possible implications in the management of OP poisoning., (Copyright © 2019 Elsevier Inc. All rights reserved.)