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Nematode parasites of humans and livestock pose a significant burden to human health, economic development, and food security. Anthelmintic drug resistance is widespread among parasites of livestock and many nematode parasites of humans lack effective treatments. Here, we present a nitrophenyl-piperazine scaffold that induces motor defects rapidly in the model nematode Caenorhabditis elegans. We call this scaffold Nemacol and show that it inhibits the vesicular acetylcholine transporter (VAChT), a target recognized by commercial animal and crop health groups as a viable anthelmintic target. We demonstrate that it is possible to create Nemacol analogs that maintain potent in vivo activity whilst lowering their affinity to the mammalian VAChT 10-fold. We also show that Nemacol enhances the ability of the anthelmintic Ivermectin to paralyze C. elegans and the ruminant nematode parasite Haemonchus contortus. Hence, Nemacol represents a promising new anthelmintic scaffold that acts through an identified viable anthelmintic target.

The association between oxidative processes and physiological responses has received much attention in ecotoxicity assessment. In the Baltic Sea, bloom-forming cyanobacterium Nodularia spumigena is a significant producer of various bioactive compounds, and both positive and adverse effects on grazers feeding in cyanobacteria blooms are reported. To elucidate the effect mechanisms and species sensitivity to the cyanobacteria-dominating diet, we exposed two Baltic copepods, Acartia bifilosa and Eurytemora affinis, to a diet consisting of toxin-producing cyanobacteria N. spumigena and a high-quality food Rhodomonas salina at 0-300 mu g C L-1; the control food was R. salina provided as a monodiet at the same food levels. The subcellular responses to food type and availability were assayed using a suite of biomarkers - antioxidant enzymes [superoxide dismutases (SOD), catalase (CAT), and glutathione S-transferases (GST)] and acetylcholinesterase (AChE). In parallel, we measured feeding activity using gut content (GC) assayed by real-time PCR analysis that quantified amounts of the prey DNA in copepod stomachs. As growth and reproduction endpoints, individual RNA content (a proxy for protein synthesis capacity), egg production rate (EPR), and egg viability (EV%) were used. In both toxic and nontoxic foods, copepod GC, RNA content, and EPR increased with food availability. Antioxidant enzyme activities increased with food availability regardless of the diet type. Moreover, CAT (both copepods), SOD, and GST (A. bifilosa) were upregulated in the copepods receiving cyanobacteria; the response was detectable when adjusted for the feeding and/or growth responses. By contrast, the diet effects were not significant when food concentration was used as a co-variable. A bimodal response in AChE was observed in A. bifilosa feeding on cyanobacteria, with up to 52% increase at the lower levels (5-25 mu g C L-1) and 32% inhibition at the highest food concentrations. These findings contr

Concerned by the urgent need to explore new approaches for the treatment of Alzheimer's disease, we herein describe the synthesis and evaluation of new multitarget molecules. In particular, we have focused our attention on modulating the activity of cholinesterases (AChE, BuChE) in order to restore the levels of the neurotransmitter acetylcholine, and of O-GlcNAcase (OGA), which is associated with hyperphosphorylation of tau protein, in turn related to the formation of neurofibrillary tangles in the brain. Specifically, we considered the possibility of using carbohydrate-fused 1,3-selenazolines, decorated with a 2-alkylamino or 2-alkoxy moieties. On the one hand, the presence of a selenium atom might be useful in modulating the intrinsic oxidative stress in AD. On the other hand, such bicyclic structure might behave as a transition state analogue of OGA hydrolysis. Moreover, upon protonation, it could mimic the ammonium cation of acetylcholine. The lead compound, bearing a propylamino moiety on C-2 position of the selenazoline motif, proved to be a good candidate against AD; it turned out to be a strong inhibitor of BuChE (IC50 = 0.46 µM), the most prevalent cholinesterase in advanced disease stages, with a roughly 4.8 selectivity index in connection to AChE (IC50 = 2.2 µM). This compound exhibited a roughly 12-fold increase in activity compared to galantamine, one of the currently marketed drugs against AD, and a selective AChE inhibitor, and virtually the same activity as rivastigmine, a selective BuChE inhibitor. Furthermore, it was also endowed with a strong inhibitory activity against human OGA, within the nanomolar range (IC50 = 0.053 µM for hOGA, >100 µM for hHexB), and, thus, with an outstanding selectivity (IC50(hHexB)/IC50(hOGA) > 1887). The title compounds also exhibited an excellent selectivity against a panel of glycosidases and a negligible cytotoxicity against tumor and non-tumor cell lines. Docking simulations performed on the three target enzymes (A

Organophosphate compounds (OPs) are highly acute toxic compounds used in agriculture production as pesticides. They bind to acetylcholinesterase leading to nerve paralysis and organ failure in humans. Hence, it has become mandatory on a global level to detect and remove organophosphate compounds from the environment. There are many conventional methods (nanoscale materials or sophisticated instruments) available to detect these compounds. However, these methods are time-consuming, expensive, and require an analytical laboratory with skilled staff. These constraints were the driving force for developing sensing platforms capable of detecting pesticides with excellent sensitivity. Metal-organic frameworks (MOFs) belong to porous coordination polymers fabricated from metal clusters/ions and multifunctional organic ligands. The tunable size and shape of pores, stable framework and the opportunity for desirable post-synthetic modifications classify them as promising candidates for potential use in detecting OPs at low nanomolar levels. This chapter will discuss the application of MOFs for the detection of selected organophosphate compounds. The special emphasis will be on MOF-based sensors, their sensitivity, selectivity, advantages and disadvantages in the OPs detection process. © 2022 Nova Science Publishers, Inc.

Organophosphate-based pesticides have remarkably contributed to the agriculture industry, but their toxicity has a large negative impact on the environment as well as on the health of humans and other living organisms. Most of the methods developed to remedy the organophosphate pesticide toxicity are very time-consuming and are based on their adsorption onto different materials and/or their degradation to nontoxic species. In this study, detoxification of three structurally different organophosphate pesticides was investigated using an NU-1000 metal–organic framework. We showed that NU-1000 is an excellent agent for fast (average time ≤ 3 min) and effective removal of organophosphate pesticides with an aromatic heterocyclic moiety. In particular, superior detoxification of chlorpyrifos solution after NU-1000 treatment was achieved after only 1 min. The combination of experimental and computational methods revealed that the synergic effects of sorption and hydrolysis are responsible for the superior removal of CHP by NU-1000. The sorption process occurs on the Zr node (chemisorption) and pyrene linkers (physisorption) following pseudo-first-order kinetics during the first minute, and a pseudo-second-order model fits the entire time range. The multilayer adsorption of chlorpyrifos or its hydrolyzed product, 3,5,6-trichloro-2-pyridinol, takes place on a pyrene linker, whereas the aliphatic part of the molecule remains chemisorbed on the Zr node. Such unique synergy between induced sorption and hydrolysis of chlorpyrifos by NU-1000 results in its fast and effective removal with rapid detoxification in non-buffered solutions.

A series of 14 target benzyl [2-(arylsulfamoyl)-1-substituted-ethyl]carbamates was prepared by multi-step synthesis and characterized. All the final compounds were tested for their ability to inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in vitro, and the selectivity index (SI) was determined. Except for three compounds, all compounds showed strong preferential inhibition of BChE, and nine compounds were even more active than the clinically used rivastigmine. Benzyl {(2S)-1-[(2-methoxybenzyl)sulfamoyl]-4-methylpentan-2-yl}carbamate, benzyl {(2S)-1-[(4-chlorobenzyl)sulfamoyl]-4-methylpentan-2-yl}carbamate, and benzyl [(2S)-1-(benzylsulfamoyl)-4-methylpentan-2-yl]carbamate showed the highest BChE inhibition (IC50 = 4.33, 6.57, and 8.52 mu M, respectively), indicating that the last two derivatives had approximately 5-fold higher inhibitory activity against BChE than rivastigmine, and the first one was even 9-fold more effective than rivastigmine. In addition, the selectivity index was approx. 10 or 34, respectively. The process of carbamylation and reactivation of BChE was studied for the most active derivatives 5k, 5j. The detailed information about the mode of binding of these compounds to the active site of both BChE and AChE was obtained in a molecular modeling study. In this study, combined techniques (docking, molecular dynamic simulations, and QTAIM (quantum theory of atoms in molecules) calculations) were employed., Vícestupňovou syntézou byla připravena a následně charakterizována série 14 cílových benzyl [2-(arylsulfamoyl)-1-substituovaných-ethyl]karbamátů. Všechny finální sloučeniny byly testovány na jejich schopnost inhibovat acetylcholinesterázu (AChE) a butyrylcholinesterázu (BChE) in vitro a byl stanoven index selektivity (SI). Kromě tří sloučenin všechny sloučeniny vykazovaly silnou preferenční inhibici BChE a devět sloučenin bylo dokonce aktivnějších než klinicky používaný rivastigmin. Benzyl {(2S)-1-[(2-methoxybenzyl)sulfamoyl]-4-methylpentan-2-yl}karbamát, benzyl {(2S)-1-[(4-chlorbenzyl)sulfamoyl]-4-methylpentan -2-yl}karbamát a benzyl [(2S)-1-(benzylsulfamoyl)-4-methylpentan-2-yl]karbamát vykazovaly nejvyšší inhibici BChE (IC50 = 4,33, 6,57 a 8,52 mu M), což ukazuje, že poslední dva deriváty měly přibližně 5krát vyšší inhibiční aktivitu proti BChE než rivastigmin a první byl dokonce 9krát účinnější než rivastigmin. Navíc jejich index selektivity byl cca. 10 respektive 34. Proces karbamylace a reaktivace BChE byl studován u nejaktivnějších derivátů. Detailní informace o způsobu vazby těchto sloučenin na aktivní místo BChE i AChE byly získány ve studii molekulárního modelování. V této studii byly použity kombinované techniky (docking, molekulárně dynamické simulace a výpočty QTAIM (kvantová teorie atomů v molekulách)).

Organophosphate-based pesticides have remarkably contributed to the agriculture industry, but their toxicity has a large negative impact on the environment as well as on the health of humans and other living organisms. Most of the methods developed to remedy the organophosphate pesticide toxicity are very time-consuming and are based on their adsorption onto different materials and/or their degradation to nontoxic species. In this study, detoxification of three structurally different organophosphate pesticides was investigated using an NU-1000 metal-organic framework. We showed that NU-1000 is an excellent agent for fast (average time ≤ 3 min) and effective removal of organophosphate pesticides with an aromatic heterocyclic moiety. In particular, superior detoxification of chlorpyrifos solution after NU-1000 treatment was achieved after only 1 min. The combination of experimental and computational methods revealed that the synergic effects of sorption and hydrolysis are responsible for the superior removal of CHP by NU-1000. The sorption process occurs on the Zr node (chemisorption) and pyrene linkers (physisorption) following pseudo-first-order kinetics during the first minute, and a pseudo-second-order model fits the entire time range. The multilayer adsorption of chlorpyrifos or its hydrolyzed product, 3,5,6-trichloro-2-pyridinol, takes place on a pyrene linker, whereas the aliphatic part of the molecule remains chemisorbed on the Zr node. Such unique synergy between induced sorption and hydrolysis of chlorpyrifos by NU-1000 results in its fast and effective removal with rapid detoxification in non-buffered solutions.

Concerned by the urgent need to explore new approaches for the treatment of Alzheimer's disease, we herein describe the synthesis and evaluation of new multitarget molecules. In particular, we have focused our attention on modulating the activity of cholinesterases (AChE, BuChE) in order to restore the levels of the neurotransmitter acetylcholine, and of O-GlcNAcase (OGA), which is associated with hyperphosphorylation of tau protein, in turn related to the formation of neurofibrillary tangles in the brain. Specifically, we considered the possibility of using carbohydrate-fused 1,3-selenazolines, decorated with a 2-alkylamino or 2-alkoxy moieties. On the one hand, the presence of a selenium atom might be useful in modulating the intrinsic oxidative stress in AD. On the other hand, such bicyclic structure might behave as a transition state analogue of OGA hydrolysis. Moreover, upon protonation, it could mimic the ammonium cation of acetylcholine. The lead compound, bearing a propylamino moiety on C-2 position of the selenazoline motif, proved to be a good candidate against AD; it turned out to be a strong inhibitor of BuChE (IC50 = 0.46 µM), the most prevalent cholinesterase in advanced disease stages, with a roughly 4.8 selectivity index in connection to AChE (IC50 = 2.2 µM). This compound exhibited a roughly 12-fold increase in activity compared to galantamine, one of the currently marketed drugs against AD, and a selective AChE inhibitor, and virtually the same activity as rivastigmine, a selective BuChE inhibitor. Furthermore, it was also endowed with a strong inhibitory activity against human OGA, within the nanomolar range (IC50 = 0.053 µM for hOGA, >100 µM for hHexB), and, thus, with an outstanding selectivity (IC50(hHexB)/IC50(hOGA) > 1887). The title compounds also exhibited an excellent selectivity against a panel of glycosidases and a negligible cytotoxicity against tumor and non-tumor cell lines. Docking simulations performed on the three target enzymes (A

CRUE-CSIC (Acuerdos Transformativos 2021), Cadmium (Cd) single and repeated exposure produces cognitive dysfunctions. Basal forebrain cholinergic neurons (BFCN) regulate cognitive functions. BFCN loss or cholinergic neurotransmission dysfunction leads to cognitive disabilities. Thyroid hormones (THs) maintain BFCN viability and functions, and Cd disrupts their levels. However, Cd-induced BFCN damages and THs disruption involvement was not studied. To research this we treated male Wistar rats intraperitoneally with Cd once (1 mg/kg) or repetitively for 28 days (0.1 mg/kg) with/without triiodothyronine (T3, 40 µg/kg/day). Cd increased thyroid-stimulating-hormone (TSH) and decreased T3 and tetraiodothyronine (T4). Cd altered cholinergic transmission and induced a more pronounced neurodegeneration on BFCN, mediated partially by THs reduction. Additionally, Cd antagonized muscarinic 1 receptor (M1R), overexpressed acetylcholinesterase S variant (AChE-S), downregulated AChE-R, M2R, M3R and M4R, and reduced AChE and choline acetyltransferase activities through THs disruption. These results may assist to discover cadmium mechanisms that induce cognitive disabilities, revealing a new possible therapeutic tool., Universidad Complutense de Madrid/Banco de Santander, Depto. de Química en Ciencias Farmacéuticas, Depto. de Medicina Legal, Psiquiatría y Patología, Sección Deptal. de Farmacología y Toxicología (Veterinaria), Fac. de Farmacia, Fac. de Medicina, Fac. de Veterinaria, TRUE, pub

Preclinical efforts to improve medical countermeasures against organophosphate (OP) chemical threat agents have largely focused on adult male models. However, age and sex have been shown to influence the neurotoxicity of repeated low-level OP exposure. Therefore, to determine the influence of sex and age on outcomes associated with acute OP intoxication, postnatal day 28 Sprague-Dawley male and female rats were exposed to the OP diisopropylfluorophosphate (DFP; 3.4 mg/kg, s.c.) or an equal volume of vehicle (∼80 µL saline, s.c.) followed by atropine sulfate (0.1 mg/kg, i.m.) and pralidoxime (2-PAM; 25 mg/kg, i.m.). Seizure activity was assessed during the first 4 h post-exposure using behavioral criteria and electroencephalographic (EEG) recordings. At 1 d post-exposure, acetylcholinesterase (AChE) activity was measured in cortical tissue, and at 1, 7, and 28 d post-exposure, brains were collected for neuropathologic analyses. At 1 month post-DFP, animals were analyzed for motor ability, learning and memory, and hippocampal neurogenesis. Acute DFP intoxication triggered more severe seizure behavior in males than females, which was supported by EEG recordings. DFP caused significant neurodegeneration and persistent microglial activation in numerous brain regions of both sexes, but astrogliosis occurred earlier and was more severe in males compared to females. DFP males and females exhibited pronounced memory deficits relative to sex-matched controls. In contrast, acute DFP intoxication altered hippocampal neurogenesis in males, but not females. These findings demonstrate that acute DFP intoxication triggers seizures in juvenile rats of both sexes, but the seizure severity varies by sex. Some, but not all, chronic neurotoxic outcomes also varied by sex. The spatiotemporal patterns of neurological damage suggest that microglial activation may be a more important factor than astrogliosis or altered neurogenesis in the pathogenesis of c

Preclinical efforts to improve medical countermeasures against organophosphate (OP) chemical threat agents have largely focused on adult male models. However, age and sex have been shown to influence the neurotoxicity of repeated low-level OP exposure. Therefore, to determine the influence of sex and age on outcomes associated with acute OP intoxication, postnatal day 28 Sprague-Dawley male and female rats were exposed to the OP diisopropylfluorophosphate (DFP; 3.4 mg/kg, s.c.) or an equal volume of vehicle (∼80 µL saline, s.c.) followed by atropine sulfate (0.1 mg/kg, i.m.) and pralidoxime (2-PAM; 25 mg/kg, i.m.). Seizure activity was assessed during the first 4 h post-exposure using behavioral criteria and electroencephalographic (EEG) recordings. At 1 d post-exposure, acetylcholinesterase (AChE) activity was measured in cortical tissue, and at 1, 7, and 28 d post-exposure, brains were collected for neuropathologic analyses. At 1 month post-DFP, animals were analyzed for motor ability, learning and memory, and hippocampal neurogenesis. Acute DFP intoxication triggered more severe seizure behavior in males than females, which was supported by EEG recordings. DFP caused significant neurodegeneration and persistent microglial activation in numerous brain regions of both sexes, but astrogliosis occurred earlier and was more severe in males compared to females. DFP males and females exhibited pronounced memory deficits relative to sex-matched controls. In contrast, acute DFP intoxication altered hippocampal neurogenesis in males, but not females. These findings demonstrate that acute DFP intoxication triggers seizures in juvenile rats of both sexes, but the seizure severity varies by sex. Some, but not all, chronic neurotoxic outcomes also varied by sex. The spatiotemporal patterns of neurological damage suggest that microglial activation may be a more important factor than astrogliosis or altered neurogenesis in the pathogenesis of c

Preclinical efforts to improve medical countermeasures against organophosphate (OP) chemical threat agents have largely focused on adult male models. However, age and sex have been shown to influence the neurotoxicity of repeated low-level OP exposure. Therefore, to determine the influence of sex and age on outcomes associated with acute OP intoxication, postnatal day 28 Sprague-Dawley male and female rats were exposed to the OP diisopropylfluorophosphate (DFP; 3.4 mg/kg, s.c.) or an equal volume of vehicle (∼80 µL saline, s.c.) followed by atropine sulfate (0.1 mg/kg, i.m.) and pralidoxime (2-PAM; 25 mg/kg, i.m.). Seizure activity was assessed during the first 4 h post-exposure using behavioral criteria and electroencephalographic (EEG) recordings. At 1 d post-exposure, acetylcholinesterase (AChE) activity was measured in cortical tissue, and at 1, 7, and 28 d post-exposure, brains were collected for neuropathologic analyses. At 1 month post-DFP, animals were analyzed for motor ability, learning and memory, and hippocampal neurogenesis. Acute DFP intoxication triggered more severe seizure behavior in males than females, which was supported by EEG recordings. DFP caused significant neurodegeneration and persistent microglial activation in numerous brain regions of both sexes, but astrogliosis occurred earlier and was more severe in males compared to females. DFP males and females exhibited pronounced memory deficits relative to sex-matched controls. In contrast, acute DFP intoxication altered hippocampal neurogenesis in males, but not females. These findings demonstrate that acute DFP intoxication triggers seizures in juvenile rats of both sexes, but the seizure severity varies by sex. Some, but not all, chronic neurotoxic outcomes also varied by sex. The spatiotemporal patterns of neurological damage suggest that microglial activation may be a more important factor than astrogliosis or altered neurogenesis in the pathogenesis of c

Objective: to review studies on the prevalence and factors related to pain in Brazilian university students.Methods: systematic review with PROSPERO record (CRD42020204197), of articles published in national and international journals, in PUBMED, EBSCO, LILACS, MEDLINE, VHL Portal, Google Scholar and SciELO. Descriptors: “Pain”, “Chronic Pain”, Students “,” Students, Health Occupations “and” Universities “. Included: a) Observational studies; b) transversal; c) published in national or international journals; d) written in English or Portuguese; e) developed with academics, in Brazilian higher education institutions; f) who have assessed the prevalence and factors related to pain; g) Such studies should be available in full. There were no restrictions on the period of publication of the studies. Excluded: h) studies that did not report the methodology applied to measure the outcome; i) studies with instruments that did not evaluate pain as a primary outcome, subsequently presenting insufficient data to analyze the results; j) studies with academics from other countries and k) studies with inconsistent data related to the sample and its main results. The risk of bias was assessed using the Downs and Black scale and proposed by Hoy.Results: searches identified 67 articles, however, after analysis 10 were included. These were cross-sectional studies, published between 2011 and 2019, 5 of them from the Northeast region. The sample totaled 3,268 students, 68% of whom were women. The prevalence of pain varied between 14.4% and 98% and chronic pain between 11.5% and 59.7%. The highest perception of self-reported pain was 4.12 ± 2.15. The main pain complaints were in the lower back and upper limbs. In the methodological analysis, studies have a moderate to high risk of bias.Conclusions: finally, the evidence indicates a high prevalence of pain, as well as its chronicity in university students. However, studies with adequate methodological rigor are still needed to confirm t, Objetivo: revisar estudos sobre a prevalência e os fatores relacionados a dor em estudantes universitários brasileiros.Métodos: revisão sistemática com registro na Prospero (CRD42020204197), de artigos publicados em periódicos nacionais e internacionais, nas bases Pubmed, Ebsco, Lilacs, Medline, Portal da BVS, Google Acadêmico e SciELO. Descritores: “Pain”, “Chronic Pain”, Students”, “Students, Health Occupations” e “Universities”. Incluídos: a) estudos observacionais; b) transversais; c) publicados em periódicos nacionais ou internacionais; d) redigidos em inglês ou português; e) desenvolvidos com acadêmicos, em instituições de ensino superior brasileiras; f) que tenham avaliado a prevalência e fatores relacionados a dor; g) Tais estudos deviam estar disponíveis na íntegra. Não foram realizadas restrições quanto ao período de publicação dos estudos. Excluídos: h) estudos que não relataram a metodologia aplicada para mensuração do desfecho; i) estudos com instrumentos que não avaliaram a dor como desfecho primário, posteriormente apresentando dados insuficientes para análise dos resultados; j) estudos com acadêmicos de outros países; e k) estudos com inconsistência dos dados relacionados a amostra e seus principais resultados. O risco de viés foi avaliado com a escala Downs and Black e a proposta por Hoy.Resultados: as buscas identificaram 67 artigos, contudo, após análise, 10 foram incluídos. Esses eram estudos transversais, publicados entre 2011 e 2019, sendo cinco deles da região Nordeste. A amostra totalizou 3.268 acadêmicos, sendo 68% mulheres. A prevalência da dor variou entre 14,4% e 98% e a dor crônica entre 11,5% e 59,7%. A maior percepção da dor autorrelatada foi de 4,12 ± 2,15. As principais queixas álgicas foram nas regiões de lombar e de membros superiores. Na análise metodológica, os estudos possuem moderado a alto risco de viés.Conclusões: por fim, as evidências indicam uma alta prevalência de dor, bem como sua cronificação em universitários. Contudo

Three coordination compounds featuring different types of tetracopper(II) cores, namely [O⊂Cu4{N(CH2CH2O)3}4(BOH)4][BF4]2 (1), [Cu4(μ4-H2edte)(μ5-H2edte)(sal)2]n·7nH2O, (H4edte=N,N,N′, N′-tetrakis(2-hydroxyethyl)ethylenediamine, H2sal=salicylic acid) (2), and [{Cu4(μ3-Hbes)4(μ-hba)}K (H2O)3]n, H3bes=N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (3), were assayed for their potency to inhibit the acetyl (AChE) and butyrylcholinesterase (BuChE) enzymes aiming to test these compounds as potential dual inhibitors in the treatment of Alzheimer's disease. All the investigated compounds showed a strong inhibitory potency toward both enzymes with IC50 values in micromolar range of concentration; compound 1 displayed the most potent inhibitory behaviour toward both enzymes. The mechanism of the AChE and BuChE inhibition was examined by enzyme kinetic measurements. The obtained kinetic parameters, Vmax and Km indicated an uncompetitive type of inhibition of both enzymes by compound 1. For the other two compounds a non-competitive inhibition mode was observed. To get further insight into the mechanism of action and to elucidate binding modes in details we examined the interactions of 1–3 with acetylcholinesterase, using molecular docking approach. Grid based docking studies indicated that these compounds can bind to peripheral anionic site (PAS) of the AChE with Ki values in micromolar range. Moreover, blind docking revealed the capability of investigated compounds to bind to new allosteric site (i.e. binding site II) distinct from PAS. Showing that these Cu-based compounds can act as new allosteric inhibitors of AChE and identifying novel allosteric binding site on AChE represents a significant contribution toward the design of novel and more effective inhibitors of AChE.

Acetylcholine (ACh) regulates inflammation via α7 nicotinic acetylcholine receptor (α7 nAChR). Acetylcholinesterase (AChE), an enzyme hydrolyzing ACh, is expressed in immune cells suggesting non-classical function in inflammatory responses. Here, the expression of PRiMA-linked G4 AChE was identified on the surface of macrophages. In lipopolysaccharide-induced inflammatory processes, AChE was upregulated by the binding of NF-κB onto the ACHE promotor. Conversely, the overexpression of G4 AChE inhibited ACh-suppressed cytokine release and cell migration, which was in contrast to that of applied AChE inhibitors. AChEmt, a DNA construct without enzymatic activity, was adopted to identify the protein role of AChE in immune system. Overexpression of G4 AChEmt induced cell migration and inhibited ACh-suppressed cell migration. The co-localization of α7 nAChR and AChE was found in macrophages, suggesting the potential interaction of α7 nAChR and AChE. Besides, immunoprecipitation showed a close association of α7 nAChR and AChE protein in cell membrane. Hence, the novel function of AChE in macrophage by interacting with α7 nAChR was determined. Together with hydrolysis of ACh, AChE plays a direct role in the regulation of inflammatory response. As such, AChE could serve as a novel target to treat age-related diseases by anti-inflammatory responses. © 2020 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences

Three coordination compounds featuring different types of tetracopper(II) cores, namely [O ⊂ Cu4N(CH2CH2O)34(BOH)4][BF4]2 (1), [Cu4(μ4-H2edte)(μ5-H2edte)(sal)2]n·7nH2O, (H4edte = N,N,N′,N′-tetrakis(2-hydroxyethyl)ethylenediamine, H2sal = salicylic acid) (2), and [Cu4(μ3-Hbes)4(μ-hba)K(H2O)3]n, H3bes = N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (3), were assayed for their potency to inhibit the acetyl (AChE) and butyrylcholinesterase (BuChE) enzymes aiming to test these compounds as potential dual inhibitors in the treatment of Alzheimer's disease. All the investigated compounds showed a strong inhibitory potency toward both enzymes with IC50 values in micromolar range of concentration; compound 1 displayed the most potent inhibitory behaviour toward both enzymes. The mechanism of the AChE and BuChE inhibition was examined by enzyme kinetic measurements. The obtained kinetic parameters, Vmax and Km indicated an uncompetitive type of inhibition of both enzymes by compound 1. For the other two compounds a non-competitive inhibition mode was observed. To get further insight into the mechanism of action and to elucidate binding modes in details we examined the interactions of 1–3 with acetylcholinesterase, using molecular docking approach. Grid based docking studies indicated that these compounds can bind to peripheral anionic site (PAS) of the AChE with Ki values in micromolar range. Moreover, blind docking revealed the capability of investigated compounds to bind to new allosteric site (i.e. binding site II) distinct from PAS. Showing that these Cu-based compounds can act as new allosteric inhibitors of AChE and identifying novel allosteric binding site on AChE represents a significant contribution toward the design of novel and more effective inhibitors of AChE. © 2020 Elsevier Inc.

Acetylcholinesterase (AChE) inhibitors are important in the treatment of neurodegenerative diseases. Two inhibitors, 12-tungstosilicic acid (WSiA) and 12-tungstophosphoric acid (WPA), which have polyoxometalate (POM) type structure, have been shown to inhibit AChE activity in nM concentration. Circular dichroism and tryptophan fluorescence spectroscopy demonstrated that the AChE inhibition was not accompanied by significant changes in the secondary structure of the enzyme. The molecular docking approach has revealed a new allosteric binding site, termed β-allosteric site (β-AS), which is considered responsible for the inhibition of AChE by POMs. To the best of our knowledge, this is the first study reporting a new allosteric site that is considered responsible for AChE inhibition by voluminous and negatively charged molecules such as POMs. The selected POMs were further subjected to genotoxicity testing using human peripheral blood cells as a model system. It was shown that WSiA and WPA induced a mild cytostatic but not genotoxic effects in human lymphocytes, which indicates their potential to be used as medicinal drugs. The identification of non-toxic compounds capable of binding to an allosteric site that so far has not been considered responsible for enzyme inhibition could be fundamental for the development of new drug design strategies and the discovery of more efficient AChE modulators.

Acetylcholinesterase (AChE) inhibitors are important in the treatment of neurodegenerative diseases. Two inhibitors,12-tungstosilicic acid (WSiA) and 12-tungstophosphoric acid (WPA), which have polyoxometalate(POM) type structure, have been shown to inhibit AChE activity in nM concentration. Circular dichroism andtryptophan fluorescence spectroscopy demonstrated that the AChE inhibition was not accompanied by significantchanges in the secondary structure of the enzyme. The molecular docking approach has revealed a newallosteric binding site, termed β-allosteric site (β-AS), which is considered responsible for the inhibition of AChEby POMs. To the best of our knowledge, this is the first study reporting a new allosteric site that is consideredresponsible for AChE inhibition by voluminous and negatively charged molecules such as POMs. The selectedPOMs were further subjected to genotoxicity testing using human peripheral blood cells as a model system. Itwas shown that WSiA and WPA induced a mild cytostatic but not genotoxic effects in human lymphocytes, whichindicates their potential to be used as medicinal drugs. The identification of non-toxic compounds capable ofbinding to an allosteric site that so far has not been considered responsible for enzyme inhibition could befundamental for the development of new drug design strategies and the discovery of more efficient AChEmodulators.

The military policeman's work activity can lead to musculoskeletal overload, due to long working hours, standing for many hours and the use of heavy equipment such as the ballistic vest, which can cause discomfort, fatigue and musculoskeletal pain, predisposing these professionals to pain. That compromise your health and quality of life. This study aimed to evaluate the perception of musculoskeletal pain of military police, identifying the presence of painful points and establishing relationships between the points found and their working conditions. Fifty-five military police officers of both sexes, with more than five years of service to the institution, who belonged to the 15th Battalion of the city of Patos de Minas-MG, participated in the study. The sociodemographic and health questionnaire was applied and, subsequently, the “Body Pain Map”. All police officers had pain in some region of the body after the workday. Associations between the body regions with pain, the use of the ballistic vest, work vehicle and length of service, showed involvement mainly in the lower back region. Given the results presented, there is a prevalence of pain in the lower back followed by pain in the thoracic region. All participants were wearing the ballistic vest, a fact that influenced the presence of the painful points presented. Therefore, studies are important in order to detect health risk factors for these workers, in order to propose strategies that improve their quality of life., La actividad laboral de la policía militar puede derivar en sobrecarga musculoesquelética, debido a las largas jornadas laborales, de pie durante muchas horas y al uso de equipo pesado como chaleco balístico, que puede generar malestar, fatiga y dolor musculoesquelético, predisponiendo a estos profesionales al dolor que comprometen su salud y calidad de vida. Este estudio tuvo como objetivo evaluar la percepción del dolor musculoesquelético por parte de la policía militar, identificando la presencia de puntos sensibles y estableciendo relaciones entre los puntos encontrados y sus condiciones de trabajo. En el estudio participaron 55 policías militares, de ambos sexos, con más de cinco años de servicio en la institución, que pertenecían a la guarnición del 15º Batallón de la ciudad de Patos de Minas-MG. Se aplicó el cuestionario sociodemográfico y de salud y, posteriormente, el “Mapa corporal del dolor”. Todos los policías presentaron dolor en alguna región del cuerpo luego de la jornada laboral. Las asociaciones entre regiones corporales con dolor, uso de chaleco balístico, vehículo de trabajo y tiempo de servicio, mostraron afectación principalmente en la región lumbar. A la vista de los resultados presentados, existe una prevalencia de dolor en la región lumbar seguido de dolor en la región torácica. Todos los participantes usaron chaleco balístico, hecho que influyó en la presencia de los puntos dolorosos presentados. Por tanto, es importante realizar estudios con el fin de detectar factores de riesgo para la salud de estos trabajadores, con el fin de proponer estrategias que mejoren su calidad de vida., A atividade laboral do policial militar pode levar à sobrecarga osteomuscular, devido a jornada de trabalho extensa, permanência por muitas horas em pé e uso de equipamentos pesados como o colete balístico, que podem gerar desconforto, fadiga e dor musculoesquelética, predispondo esses profissionais a dores que comprometem sua saúde e qualidade de vida. Este estudo teve como objetivo avaliar a percepção de dor musculoesquelética de policiais militares, identificando a presença de pontos dolorosos e estabelecendo relações entre os pontos encontrados e suas condições de trabalho. Participaram do estudo 55 policiais militares, de ambos os sexos, com mais de cinco anos de serviço prestado à instituição, que pertenciam à guarnição do 15° Batalhão da cidade de Patos de Minas-MG. Foi aplicado o questionário sociodemográfico e de saúde e, posteriormente, o “Mapa Corporal da Dor”. Todos os policiais apresentaram dor em alguma região do corpo após a jornada de trabalho. Associações entre as regiões corporais com dor, o uso do colete balístico, veículo de trabalho e tempo de serviço prestado, evidenciaram acometimento principalmente na região das costas inferior. Diante dos resultados apresentados, observa-se uma prevalência de dores na região lombar seguida de dores na região torácica. Todos os participantes faziam uso do colete balístico, fato que influenciou a presença dos pontos dolorosos apresentados. Portanto, faz-se importante a realização de estudos, com intuito de detectar fatores de risco à saúde desses trabalhadores, a fim de propor estratégias que melhorem sua qualidade de vida.

The military policeman's work activity can lead to musculoskeletal overload, due to long working hours, standing for many hours and the use of heavy equipment such as the ballistic vest, which can cause discomfort, fatigue and musculoskeletal pain, predisposing these professionals to pain. That compromise your health and quality of life. This study aimed to evaluate the perception of musculoskeletal pain of military police, identifying the presence of painful points and establishing relationships between the points found and their working conditions. Fifty-five military police officers of both sexes, with more than five years of service to the institution, who belonged to the 15th Battalion of the city of Patos de Minas-MG, participated in the study. The sociodemographic and health questionnaire was applied and, subsequently, the “Body Pain Map”. All police officers had pain in some region of the body after the workday. Associations between the body regions with pain, the use of the ballistic vest, work vehicle and length of service, showed involvement mainly in the lower back region. Given the results presented, there is a prevalence of pain in the lower back followed by pain in the thoracic region. All participants were wearing the ballistic vest, a fact that influenced the presence of the painful points presented. Therefore, studies are important in order to detect health risk factors for these workers, in order to propose strategies that improve their quality of life., La actividad laboral de la policía militar puede derivar en sobrecarga musculoesquelética, debido a las largas jornadas laborales, de pie durante muchas horas y al uso de equipo pesado como chaleco balístico, que puede generar malestar, fatiga y dolor musculoesquelético, predisponiendo a estos profesionales al dolor que comprometen su salud y calidad de vida. Este estudio tuvo como objetivo evaluar la percepción del dolor musculoesquelético por parte de la policía militar, identificando la presencia de puntos sensibles y estableciendo relaciones entre los puntos encontrados y sus condiciones de trabajo. En el estudio participaron 55 policías militares, de ambos sexos, con más de cinco años de servicio en la institución, que pertenecían a la guarnición del 15º Batallón de la ciudad de Patos de Minas-MG. Se aplicó el cuestionario sociodemográfico y de salud y, posteriormente, el “Mapa corporal del dolor”. Todos los policías presentaron dolor en alguna región del cuerpo luego de la jornada laboral. Las asociaciones entre regiones corporales con dolor, uso de chaleco balístico, vehículo de trabajo y tiempo de servicio, mostraron afectación principalmente en la región lumbar. A la vista de los resultados presentados, existe una prevalencia de dolor en la región lumbar seguido de dolor en la región torácica. Todos los participantes usaron chaleco balístico, hecho que influyó en la presencia de los puntos dolorosos presentados. Por tanto, es importante realizar estudios con el fin de detectar factores de riesgo para la salud de estos trabajadores, con el fin de proponer estrategias que mejoren su calidad de vida., A atividade laboral do policial militar pode levar à sobrecarga osteomuscular, devido a jornada de trabalho extensa, permanência por muitas horas em pé e uso de equipamentos pesados como o colete balístico, que podem gerar desconforto, fadiga e dor musculoesquelética, predispondo esses profissionais a dores que comprometem sua saúde e qualidade de vida. Este estudo teve como objetivo avaliar a percepção de dor musculoesquelética de policiais militares, identificando a presença de pontos dolorosos e estabelecendo relações entre os pontos encontrados e suas condições de trabalho. Participaram do estudo 55 policiais militares, de ambos os sexos, com mais de cinco anos de serviço prestado à instituição, que pertenciam à guarnição do 15° Batalhão da cidade de Patos de Minas-MG. Foi aplicado o questionário sociodemográfico e de saúde e, posteriormente, o “Mapa Corporal da Dor”. Todos os policiais apresentaram dor em alguma região do corpo após a jornada de trabalho. Associações entre as regiões corporais com dor, o uso do colete balístico, veículo de trabalho e tempo de serviço prestado, evidenciaram acometimento principalmente na região das costas inferior. Diante dos resultados apresentados, observa-se uma prevalência de dores na região lombar seguida de dores na região torácica. Todos os participantes faziam uso do colete balístico, fato que influenciou a presença dos pontos dolorosos apresentados. Portanto, faz-se importante a realização de estudos, com intuito de detectar fatores de risco à saúde desses trabalhadores, a fim de propor estratégias que melhorem sua qualidade de vida.

Genistein, 4′,5,7-trihydroxyisoflavone, is a major isoflavone in soybean, which is known as phytestrogen having known benefit to brain functions. Being a common phytestrogen, the possible role of genistein in the brain protection needs to be further explored. In cultured PC12 cells, application of genistein significantly induced the expression of neurofilaments (NFs), markers for neuronal differentiation. In parallel, the expression of tetrameric form of proline-rich membrane anchor (PRiMA)-linked acetylcholinesterase (G4 AChE), a key enzyme to hydrolyze acetylcholine in cholinergic synapses, was induced in a dose-dependent manner: this induction included the associated protein PRiMA. The genistein-induced AChE expression was fully blocked by the pre-treatment of H89 (an inhibitor of protein kinase A, PKA) and G15 (a selective G protein-coupled receptor 30 (GPR30) antagonist), which suggested a direct involvement of a membrane-bound estrogen receptor (ER), named as GPR30 in the cultures. In parallel, the estrogen-induced activation of GPR30 induced AChE expression in a dose-dependent manner. The genistein/estrogen-induced AChE expression was triggered by a cyclic AMP responding element (CRE) located on the ACHE gene promoter. The binding of this CRE site by cAMP response element-binding protein (CREB) induced ACHE gene transcription. In parallel, increased expression levels of miR132 and miR212 were found when cultured PC12 cells were treated with genistein or G1. Thus, a balance between production and destruction of AChE by the activation of GPR30 was reported here. We have shown for the first time that the activation of GPR30 could be one way for estrogen or flavonoids, possessing estrogenic properties, to enhance cholinergic functions in the brain, which could be a good candidate for possible treatment of neurodegenerative diseases. © 2018 Liu, Xu, Jin, Wu, Dong and Tsim.

Commercially available pesticides were examined as Mus musculus and Homo sapiens acetylcholinesterase (mAChE and hAChE) inhibitors by means of ligand-based (LB) and structure-based (SB) in silico approaches. Initially, the crystal structures of simazine, monocrotophos, dimethoate, and acetamiprid were reproduced using various force fields. Subsequently, LB alignment rules were assessed and applied to determine the inter synaptic conformations of atrazine, propazine, carbofuran, carbaryl, tebufenozide, imidacloprid, diuron, monuron, and linuron. Afterwards, molecular docking and dynamics SB studies were performed on either mAChE or hAChE, to predict the listed pesticides’ binding modes. Calculated energies of global minima (Eglob_min) and free energies of binding (∆Gbinding) were correlated with the pesticides’ acute toxicities (i.e., the LD50 values) against mice, as well to generate the model that could predict the LD50s against humans. Although for most of the pesticides the low Eglob_min correlates with the high acute toxicity, it is the ∆Gbinding that conditions the LD50 values for all the evaluated pesticides. Derived pLD50 = f(∆Gbinding) mAChE model may predict the pLD50 against hAChE, too. The hAChE inhibition by atrazine, propazine, and simazine (the most toxic pesticides) was elucidated by SB quantum mechanics (QM) DFT mechanistic and concentration-dependent kinetic studies, enriching the knowledge for design of less toxic pesticides.

Fatalities from organophosphate (OP) insecticide result from both occupational and deliberate exposure; significantly impacting human health. Like nerve agents, insecticides are neurotoxins which target and inhibit acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with a pyridinium aldoxime e.g. pralidoxime, to reactivate the OP-inhibited AChE. However, commonly used oximes inefficiently cross the bloodbrain barrier and are rapidly cleared and their benefit is debated. Recent findings have demonstrated the ability of a novel zwitterionic, centrally acting, brain penetrating oxime (RS194B) to reverse severe symptoms and rapidly reactivate sarin-inhibited AChE in macaques, but it has not been tested following OP pesticide poisoning. In the present study, the symptoms following a lethal dose of inhaled paraoxon (100ug/kg), were shown to mimic those in insecticide poisoned individuals and were also rapidly reversed in macaques by post-exposure IM administration of 80mg/kg of RS194B. This occurred with a concomitant reactivation of AChE to 40-100% in<1hr and BChE (40% in 8h). These findings will be used to develop a macaque model with RS194B as a post-exposure treatment for insecticide poisoning and generate efficacy data for approval under the FDA Animal rule.

Commercially available pesticides were examined as Mus musculus and Homo sapiens acetylcholinesterase (mAChE and hAChE) inhibitors by means of ligand-based (LB) and structure-based (SB) in silico approaches. Initially, the crystal structures of simazine, monocrotophos, dimethoate, and acetamiprid were reproduced using various force fields. Subsequently, LB alignment rules were assessed and applied to determine the inter synaptic conformations of atrazine, propazine, carbofuran, carbaryl, tebufenozide, imidacloprid, diuron, monuron, and linuron. Afterwards, molecular docking and dynamics SB studies were performed on either mAChE or hAChE, to predict the listed pesticides’ binding modes. Calculated energies of global minima (Eglob_min) and free energies of binding (∆Gbinding) were correlated with the pesticides’ acute toxicities (i.e., the LD50 values) against mice, as well to generate the model that could predict the LD50s against humans. Although for most of the pesticides the low Eglob_min correlates with the high acute toxicity, it is the ∆Gbinding that conditions the LD50 values for all the evaluated pesticides. Derived pLD50 = f(∆Gbinding) mAChE model may predict the pLD50 against hAChE, too. The hAChE inhibition by atrazine, propazine, and simazine (the most toxic pesticides) was elucidated by SB quantum mechanics (QM) DFT mechanistic and concentration-dependent kinetic studies, enriching the knowledge for design of less toxic pesticides.

Nowadays, valorization of biological wastes from agricultural activity and processing industry has become a challenge. The olive oil industry has a great economic, commercial and industrial relevance in Spain, being olive leaves one of the main by-products and a promising source of bioactive compounds. Several authors have reported the presence and biological activity of different terpenoids in olive leaves; among them, the most abundant triterpenoids in olive leaves (derived from oleanane and ursane) have been described as possessing pharmacological activity against neurodegenerative disorders. Alzheimer’s disease (AD) is the most common neurodegenerative disorder, accounting for approximately two thirds of all cases of dementia and affecting up to 20% of individuals older than 80 years. At present, there is not effective treatment for this disease. Since AD is a multifactorial neurological pathology, a current strategy is to target different factors, being one of them related to the high activity of the enzyme acetylcholinesterase (AChE) in the brain. This work was oriented towards the valorization of olive leaf residues through the systematic and selective supercritical fluid extraction (SFE) of different families of bioactive terpenoids with potential anticholinergic (anti-AChE) activity. Thus, a sequential SFE procedure was applied for the isolation of low, medium and high molecular weight terpenoids (C10-C15, C15-C20, C20-C30), using CO2 as pure solvent and different working pressures (from 80-120 bar) at 40°C. Extracts obtained at different SFE conditions were characterized by GC-MS and its anti-AChE activity evaluated. Good anti-AChE activity was obtained for some of the terpene-rich SFE extracts from olive leaves, demonstrating the interest of this strategy to help to discover promising therapeutic approaches and natural compounds that can delay the detrimental effects of AD.

The biochemical responses in muscle, such as the enzymatic activities of acetylcholinesterase, lactate dehydrogenase and isocitrate dehydrogenase, were studied in sole (Solea senegalensis) collected in Huelva estuary (SW Spain), in the vicinity of a petrochemical and mining industry. The sampling sites showed different type and degree of pollution. The results demonstrated significant differences in muscle activities of AChE and IDH in Odiel and Tinto Rivers compared to control fish. LDH activity did not show any difference between sampling sites. Significant correlations were established between some biomarkers and heavy metals: AChE was correlated with Pb, Cd and Cu concentrations in water; IDH activity was correlated with Cd and Cu concentrations in water and As, Pb and Cd concentrations in sediments; LDH activity was correlated with As and Zn concentration in water and Cd concentration in sediment. Only one correlation was established between the biomarkers analysed and the concentrations of PAHs: benzo(b)fluoranthene concentration in sediment and IDH.

The objective of this study was to highlight the nurses' performance in the management of pain in the emergency and emergency room. It was a bibliographical and descriptive research with information regarding the purpose of the study, prioritized articles published in the last ten years. The results were: Reception with risk classification in the emergency services, humanized nursing care, nursing care planning, professionals have both technical and scientific knowledge for a professional performance with efficiency and quality. Recently instruments were created with the intention of quantifying the mentioned pain, in order to improve patient care. Pain is considered especially acute pain is one of the main reasons for seeking health services, more specifically the sectors of urgency and emergency, and the pains of high intensity are those prevalent in the complaints referred by patients, O objetivo desse estudo foi destacar a atuação do enfermeiro no manejo da dor na sala de urgência e emergência. Tratou-se de uma pesquisa bibliográfica e descritiva com informações referentes ao objetivo do estudo, priorizou artigos publicados nos últimos dez anos. Os resultados foram: o acolhimento com a classificação de risco nos serviços de Urgência, a assistência de enfermagem humanizada, o planejamento da assistência de enfermagem, os profissionais possuam conhecimento tanto técnico quanto cientifico para uma atuação profissional com eficiência e qualidade. Ultimamente instrumentos foram criados com o intuito de quantificar a dor referida, na intenção de melhorar o atendimento a paciente. Considera-se a dor especialmente a dor aguda é um dos principais motivos de busca pelos serviços de saúde, mais especificamente os setores de urgência e emergência, sendo que as dores de forte intensidade são as prevalentes nas queixas referidas pelos pacientes.

The resistance levels of the Colorado potato beetle (CPB) in Serbia to organophosphorus (OP) and carbamate (C) insecticides varied by population. The resistance level of populations to OPs indescending order was: Futog, Ratari, Dobanovci and Pester. Most resistant to carbarylwere populations Dobanovci and Ratari, followed by Futog, Kaona, and Pester, respectively. The order of resistance levels for OPs and C was completely opposite. Experiments showed that acetylcholinesterase (AChE) activity of CPB was verypronounced and easily measured. At a constant AChE concentration, increasing thesubstrate concentration will cause a positive, linear and dependent increase in thereaction. The same applies in the reaction with constant substrate concentration andincreased enzyme concentrations. AChE activity is significantly affected not only bylocation but also by substrate concentration (acetylthiocholine iodide ATChI). At low substrate concentrations, the highest AChE activity was determined in population Dobanovci, followed by Kaona, Ratari and Futog, respectively. At high substrate concentrations, however, the highest AChE activity was determined in population Ratari and then in Dobanovci and Futog. The lowest rate was in population Kaona. Considering that ATChI (substrate) in increased concentrations inhibits normal AChE activity, it can be concluded that altered AChE affected the change in the populationorder. The testing of AChE in the presence of high concentrations of substrate showsthe portion of altered AChE is largest in population Ratari, and yet considerably smallerin Dobanovci, Futog and Kaona. The total AChE activity is in correlation with thedetermined resistance to carbamates.

Context: Bioprospection has become a dynamic scientific field that explores novel possibilities for the implementation of natural products in medicine and pharmacy. Compared to marine species from all kingdoms, freshwater species have been highly neglected. Objective: This work focuses on the screening of acetylcholinesterase inhibitory (AChE) and mutagenic activities of the acetone extract (obtained by maceration) of the freshwater sponge Ochridaspongia rotunda Arndt (Malawispongiidae) in vitro. Materials and methods: AChE inhibitory activity was evaluated both in liquid (five different concentrations of the extract, from 1 to 100 mu g/mL) and in solid (seven different concentrations of the extract, from 0.5 to 10.0 mu g) by methods well described in literature, while mutagenicity was estimated using the Ames test (four different concentrations of the extract, from 0.106 to 1.328mg/plate). Results: Ochridaspongia rotunda acetone extract exhibited promising AChE inhibitory activity in a dose-dependent manner both in liquid (IC50 23.07 mu g/mL) and in solid (1.50 mu g). Furthermore, the Ames test revealed no sign of mutagenicity at any concentration tested. Its FTIR spectrum coupled with the positive Liebermann-Burchard, Salkowski and Zak color reactions (tests) indicated the presence of sterol compounds. Discussion and conclusion: The screened extract may inspire a search for novel anticholinesterase therapeutic agent(s) potentially used in the treatment of Alzheimer's disease. Further research will be directed toward its detailed chemical analysis along with addressing the issue of a real producer of the natural product(s) responsible for the AChE activity observed.

Predmet istraživanja doktorske disertacije je bio usmeren na ispitivanje bioloških efekata ekstrakata korena lincure i određivanje njihovog kvalitativnog i kvantitativnog sastava. Ispitivan je uticaj ekstrakata i konstituenata lincure (G. lutea) na aktivnost enzima mijeloperoksidaze (MPO), acetilholinesteraze (AChE) i ekto-ATPaze (E-NTPDaze) sa membrane sinaptozoma. Takođe, određivana je antioksidativna, antimikrobna, radioprotektivna i antiproliferativna aktivnost ekstrakata lincure i njenih konstituenata. Najzad, ispitan je i uticaj pojedinih konstituenata lincure na proces apoptoze humanih leukocita. Za proučavanje navedenih bioloških efekata pripremljeni su vodeni, kiseli (4% sirćetna kiselina, v/v) i etanolno vodeni (25, 50, 75 i 96% etanola u vodi, v/v) ekstrakti. Genciopikrozid, loganska kiselina, sverozid, svertiamarin, izoviteksin, amarogentin i izogentizin su izabrani kao konstituenti lincure čija je biološka aktivnost ispitivana. Kvalitativni sastav ekstrakata određivan je primenom tečno-masene hromatografije: HPLC-MS/ESI-TOF, MALDI-TOF i UPLC-PDA. Kvantitativna analiza ekstrakata kojom su određivane koncentracije navedenih konstituenata izvšena je pomoću UPLCTQD- MS/MS. Kvantitativnom analizom je utvrđeno da su genciopikrozid i loganska kiselina najzastupljenija jedinjenja u svim ekstraktima (0,9 do 2,5% genciopikrozid i 0,3 do 0,9% loganska kiselina), izogentizin je prisutan samo u etanolno vodenim ekstraktima (50- 96% EtOH, v/v) a amarogentin i izoviteksin su prisutni u tragovima. Kao najpotentniji inhibitori enzima MPO, AChE i E-NTPDaze su se pokazali etanolno vodeni ekstrakti. Pri koncentraciji od 100 g/mL, 50% i 75% EtOH ekstrakti inhibiraju 100% aktivnosti MPO, a u grupi ispitivanih konstituenata najjači inhibitor je genciopikrozid sa IC50=0,80,1 g/mL (2,25 M). Najviši stepen inhibicije AChE (54%) postignut je sa 75% EtOH ekstraktom, pri koncentraciji od 50 g/mL a od konstituenata sa loganskom kiselinom (39%) pri C= 6,77 M. Sposobnost inhibic, The objective of the study of this doctoral thesis was focused toward investigation of the biological effects of G. lutea extracts and determination of their qualitative and quantitative composition. The activity of enzymes, myeloperoxidase (MPO), acetylcholinesterase (AChE) and synaptosomal membrane ecto-ATPase (ENTPDase) were investigated. Also, antioxidative, antimicrobial, radioprotective and antiproliferative activity of Gentian extracts and their constituents were determined. Finally, the influence of the individual G. lutea constituents on apoptosis of human leukocytes was examined. For the study of the mentioned biological effects water, acidic (4 % acetic acid, v/v) and ethanol-water extracts (25, 50, 75 and 96% ethanol in water, v/v) were prepared. Gentiopicroside, loganic acid, sweroside, swertiamarin, isovitexin, amarogentin and isogentisine were selected as G. lutea constituents whose biological activity was investigated. The qualitative composition of the extracts was determined by liquid chromatography coupled with mass spectrometry: HPLC-MS/ESI-TOF, MALDITOF and UPLC-PDA. Quantitative analysis of the extracts, which was used for the determination of the constituent’s concentration, was performed using UPLC-TQDMS/ MS. Quantitative analysis showed that the gentiopicroside and loganic acid are the most abundant compounds in all examined extracts (0.9 to 2.5% and 0.3-0.9% of gentiopicroside and loganic acid, respectively), while isogentisin is only present in the ethanol water extracts (50-96%, v/v) and amarogentin and isovitexin are present only in traces. The ethanol water extracts proved to be the most potent inhibitors of MPO, AChE and E-NTPDase enzymes. At a concentration of 100 μg/ml, 50% and 75% EtOH extracts inhibited 100% of MPO activity, and in the group of examined constituents the strongest inhibition was achieved with gentiopicroside with an IC50 = 0.80.1 g/mL (2.25 M). The highest degree of inhibition of AChE (54%) was obtained with 75%

The intensification of agricultural production in the Mekong Delta has faced serious challenges with respect to increased use of agrochemicals and especially pesticides. The indiscriminate use of pesticide could potentially impact on the long-term food production, environmental and human health in the delta. The aim of this thesis was to investigate the negative side effects of the current use of pesticides on climbing perch (Anabas testudineus) in rice fields using brain acetylcholinesterase (hereafter referred to as AChE) activity as a biomarker. The empirical work, on which this thesis is based, includes structured questionnaires, laboratory and field experiments. First, a field survey using questionnaires was carried out to gain a better understanding of the current state of rice farming systems, the use of pesticides and attitude to pest management strategies among rice and rice-fish farmers, as well as to provide basic information for the set-up of the laboratory and field experiments. Secondly, laboratory studies were conducted to clarify if the selected insecticides applied alone and in mixtures caused negative side effects on climbing perch fingerlings. Thirdly, further toxicity studies were carried out, under rice field conditions, to further investigate the toxicity effects of the insecticides, applied alone, in mixtures and under sequential applications, on climbing perch fingerlings. The results showed that although there were a more selective use of pesticides and an increased awareness among farmers of the negative side effects of pesticides in 2007 as compared to 1999, the current use of pesticide in the Mekong Delta still cause many problems to the environment and human health. Chlorpyrifos ethyl (hereafter referred to as CPF) was found to cause a significant and more prolonged inhibition on the brain AChE activity in climbing perch than fenobucarb (hereafter referred to as F). The inhibition by the mixture of CPF and F were significantly higher tha, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 5: Submitted.

The intensification of agricultural production in the Mekong Delta has faced serious challenges with respect to increased use of agrochemicals and especially pesticides. The indiscriminate use of pesticide could potentially impact on the long-term food production, environmental and human health in the delta. The aim of this thesis was to investigate the negative side effects of the current use of pesticides on climbing perch (Anabas testudineus) in rice fields using brain acetylcholinesterase (hereafter referred to as AChE) activity as a biomarker. The empirical work, on which this thesis is based, includes structured questionnaires, laboratory and field experiments. First, a field survey using questionnaires was carried out to gain a better understanding of the current state of rice farming systems, the use of pesticides and attitude to pest management strategies among rice and rice-fish farmers, as well as to provide basic information for the set-up of the laboratory and field experiments. Secondly, laboratory studies were conducted to clarify if the selected insecticides applied alone and in mixtures caused negative side effects on climbing perch fingerlings. Thirdly, further toxicity studies were carried out, under rice field conditions, to further investigate the toxicity effects of the insecticides, applied alone, in mixtures and under sequential applications, on climbing perch fingerlings. The results showed that although there were a more selective use of pesticides and an increased awareness among farmers of the negative side effects of pesticides in 2007 as compared to 1999, the current use of pesticide in the Mekong Delta still cause many problems to the environment and human health. Chlorpyrifos ethyl (hereafter referred to as CPF) was found to cause a significant and more prolonged inhibition on the brain AChE activity in climbing perch than fenobucarb (hereafter referred to as F). The inhibition by the mixture of CPF and F were significantly higher tha, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 5: Submitted.

Flavonoids, a group of natural compounds mainly derived from plants, are known to possess osteogenic effects in bone cells. Here, we aimed to test if flavonoid could induce a cholinergic enzyme, acetylcholinesterase (AChE), as well as bone differentiation. In cultured rat osteoblasts, twenty flavonoids, deriving from Chinese herbs and having known induction of alkaline phosphatase (ALP1) expression, were tested for its induction activity on AChE expression. Eleven flavonoids showed the induction, and five of them had robust activation of AChE expression, including baicalin, calycosin, genistin, hyperin and pratensein: the induction of AChE included the levels of mRNA, protein and enzymatic activity. Moreover, the flavonoid-induced AChE expression in cultured osteoblast was in proline-rich membrane anchor (PRiMA)-linked tetrameric globular form (G4) only. In parallel, the expression of PRiMA was also induced by the application of flavonoids. The flavonoid-induced AChE in the cultures was not affected by estrogen receptor blocker, ICI 182,780. Taken together, the induction of PRiMA-linked AChE in osteoblast should be independent to classical estrogen signaling pathway. © 2016 Elsevier Ireland Ltd

PC12 is a well studied cell model for neuronal differentiation. AChE is also considered as a marker for neuronal differentiation. In this study, we detected the change of AChE activity during the NGF induced differentiation of PC 12 cells, and targeted on the ratio of the activity of AChE on the cell surface, and found that NGF mainly increased the intracellular AChE activity. Dioxin is a kind of persistent organic pollutants which have extreme impact on human health and widely distributed all over the world. Recently, AChE was reported as a target of the toxicity of dioxin. Here we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on AChE activity in the PC12 cells, and found that at the later stage of differentiation, TCDD could decrease the AChE activity. This down regulation might not related to transcriptional regulation. © 2016 Elsevier Ireland Ltd

The expression of acetylcholinesterase (AChE), an enzyme hydrolyzes neurotransmitter acetylcholine at vertebrate neuromuscular junction, is regulated during myogenesis, indicating the significance of muscle intrinsic factors in controlling the enzyme expression. DNA methylation is essential for temporal control of myogenic gene expression during myogenesis; however, its role in AChE regulation is not known. The promoter of vertebrate ACHE gene carries highly conserved CG-rich regions, implying its likeliness to be methylated for epigenetic regulation. A DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), was applied onto C2C12 cells throughout the myotube formation. When DNA methylation was inhibited, the promoter activity, transcript expression and enzymatic activity of AChE were markedly increased after day 3 of differentiation, which indicated the putative role of DNA methylation. By bisulfite pyrosequencing, the overall methylation rate was found to peak at day 3 during C2C12 cell differentiation; a SP1 site located at -1826 bp upstream of mouse ACHE gene was revealed to be heavily methylated. The involvement of transcriptional factor SP1 in epigenetic regulation of AChE was illustrated here: (i) the SP1-driven transcriptional activity was increased in 5-Aza-treated C2C12 culture; (ii) the binding of SP1 onto the SP1 site of ACHE gene was fully blocked by the DNA methylation; and (iii) the sequence flanking SP1 sites of ACHE gene was precipitated by chromatin immuno-precipitation assay. The findings suggested the role of DNA methylation on AChE transcriptional regulation and provided insight in elucidating the DNA methylation-mediated regulatory mechanism on AChE expression during muscle differentiation. © 2016 Elsevier B.V. All rights reserved.

Adenosine 5′-triphosphate (ATP), a neurotransmitter and a neuromodulator, has been shown to be co-stored and co-released with acetylcholine (ACh) at the pre-synaptic vesicles in vertebrate neuromuscular junction (nmj). Several lines of studies demonstrated that binding of ATP to its corresponding P2Y1 receptors (P2Y1R) in muscle and neuron regulated the post-synaptic gene expressions. Indeed, the expression of acetylcholinesterase (AChE) in muscle was markedly decreased in P2Y1R−/− (P2Y1R knock-out) mice. In order to search for possible role of P2Y1R in cholinergic function of the brain, the expression of globular form AChE was determined in the brain of P2Y1R−/− mice. In contrast to that in muscle, the amounts of AChE activity, AChE catalytic subunit, structure subunit PRiMA and the amount of ACh, in the brain were not, significantly, altered, suggesting the role of P2Y1R in neuron could have different function as that in muscle. However, the expressions of a series of neuronal development markers, i.e. neurofilaments, were reduced in P2Y1R−/− mouse brain, indicating P2Y1R may be involved in neuronal development process. © 2016 Elsevier Ireland Ltd

Predmet istraživanja doktorske disertacije je bio usmeren na ispitivanjebioloških efekata ekstrakata korena lincure i određivanje njihovog kvalitativnog ikvantitativnog sastava. Ispitivan je uticaj ekstrakata i konstituenata lincure (G. lutea) naaktivnost enzima mijeloperoksidaze (MPO), acetilholinesteraze (AChE) i ekto-ATPaze(E-NTPDaze) sa membrane sinaptozoma. Takođe, određivana je antioksidativna,antimikrobna, radioprotektivna i antiproliferativna aktivnost ekstrakata lincure i njenihkonstituenata. Najzad, ispitan je i uticaj pojedinih konstituenata lincure na procesapoptoze humanih leukocita.Za proučavanje navedenih bioloških efekata pripremljeni su vodeni, kiseli (4%sirćetna kiselina, v/v) i etanolno vodeni (25, 50, 75 i 96% etanola u vodi, v/v) ekstrakti.Genciopikrozid, loganska kiselina, sverozid, svertiamarin, izoviteksin, amarogentin iizogentizin su izabrani kao konstituenti lincure čija je biološka aktivnost ispitivana.Kvalitativni sastav ekstrakata određivan je primenom tečno-masene hromatografije:HPLC-MS/ESI-TOF, MALDI-TOF i UPLC-PDA. Kvantitativna analiza ekstrakatakojom su određivane koncentracije navedenih konstituenata izvšena je pomoću UPLCTQD-MS/MS. Kvantitativnom analizom je utvrđeno da su genciopikrozid i loganskakiselina najzastupljenija jedinjenja u svim ekstraktima (0,9 do 2,5% genciopikrozid i 0,3do 0,9% loganska kiselina), izogentizin je prisutan samo u etanolno vodenimekstraktima (50- 96% EtOH, v/v) a amarogentin i izoviteksin su prisutni u tragovima.Kao najpotentniji inhibitori enzima MPO, AChE i E-NTPDaze su se pokazali etanolnovodeni ekstrakti. Pri koncentraciji od 100 g/mL, 50% i 75% EtOH ekstrakti inhibiraju100% aktivnosti MPO, a u grupi ispitivanih konstituenata najjači inhibitor jegenciopikrozid sa IC50=0,80,1 g/mL (2,25 M). Najviši stepen inhibicije AChE(54%) postignut je sa 75% EtOH ekstraktom, pri koncentraciji od 50 g/mL a odkonstituenata sa loganskom kiselinom (39%) pri C= 6,77 M. Sposobnost inhibicije ENTPDazesu pokazali, The objective of the study of this doctoral thesis was focused towardinvestigation of the biological effects of G. lutea extracts and determination of theirqualitative and quantitative composition. The activity of enzymes, myeloperoxidase(MPO), acetylcholinesterase (AChE) and synaptosomal membrane ecto-ATPase (ENTPDase)were investigated. Also, antioxidative, antimicrobial, radioprotective andantiproliferative activity of Gentian extracts and their constituents were determined.Finally, the influence of the individual G. lutea constituents on apoptosis of humanleukocytes was examined.For the study of the mentioned biological effects water, acidic (4 % acetic acid,v/v) and ethanol-water extracts (25, 50, 75 and 96% ethanol in water, v/v) wereprepared. Gentiopicroside, loganic acid, sweroside, swertiamarin, isovitexin,amarogentin and isogentisine were selected as G. lutea constituents whose biologicalactivity was investigated. The qualitative composition of the extracts was determined byliquid chromatography coupled with mass spectrometry: HPLC-MS/ESI-TOF, MALDITOFand UPLC-PDA. Quantitative analysis of the extracts, which was used for thedetermination of the constituent’s concentration, was performed using UPLC-TQDMS/MS. Quantitative analysis showed that the gentiopicroside and loganic acid are themost abundant compounds in all examined extracts (0.9 to 2.5% and 0.3-0.9% ofgentiopicroside and loganic acid, respectively), while isogentisin is only present in theethanol water extracts (50-96%, v/v) and amarogentin and isovitexin are present only intraces. The ethanol water extracts proved to be the most potent inhibitors of MPO,AChE and E-NTPDase enzymes.At a concentration of 100 μg/ml, 50% and 75% EtOH extracts inhibited 100% ofMPO activity, and in the group of examined constituents the strongest inhibition wasachieved with gentiopicroside with an IC50 = 0.80.1 g/mL (2.25 M). The highestdegree of inhibition of AChE (54%) was obtained with 75% EtOH extract at aconcentr

Flavonoids, a group of natural compounds mainly derived from plants, are known to possess osteogenic effects in bone cells. Here, we aimed to test if flavonoid could induce a cholinergic enzyme, acetylcholinesterase (AChE), as well as bone differentiation. In cultured rat osteoblasts, twenty flavonoids, deriving from Chinese herbs and having known induction of alkaline phosphatase (ALP1) expression, were tested for its induction activity on AChE expression. Eleven flavonoids showed the induction, and five of them had robust activation of AChE expression, including baicalin, calycosin, genistin, hyperin and pratensein: the induction of AChE included the levels of mRNA, protein and enzymatic activity. Moreover, the flavonoid-induced AChE expression in cultured osteoblast was in proline-rich membrane anchor (PRiMA)-linked tetrameric globular form (G4) only. In parallel, the expression of PRiMA was also induced by the application of flavonoids. The flavonoid-induced AChE in the cultures was not affected by estrogen receptor blocker, ICI 182,780. Taken together, the induction of PRiMA-linked AChE in osteoblast should be independent to classical estrogen signaling pathway. © 2016 Elsevier Ireland Ltd

PC12 is a well studied cell model for neuronal differentiation. AChE is also considered as a marker for neuronal differentiation. In this study, we detected the change of AChE activity during the NGF induced differentiation of PC 12 cells, and targeted on the ratio of the activity of AChE on the cell surface, and found that NGF mainly increased the intracellular AChE activity. Dioxin is a kind of persistent organic pollutants which have extreme impact on human health and widely distributed all over the world. Recently, AChE was reported as a target of the toxicity of dioxin. Here we investigated the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on AChE activity in the PC12 cells, and found that at the later stage of differentiation, TCDD could decrease the AChE activity. This down regulation might not related to transcriptional regulation. © 2016 Elsevier Ireland Ltd

The intensification of agricultural production in the Mekong Delta has faced serious challenges with respect to increased use of agrochemicals and especially pesticides. The indiscriminate use of pesticide could potentially impact on the long-term food production, environmental and human health in the delta. The aim of this thesis was to investigate the negative side effects of the current use of pesticides on climbing perch (Anabas testudineus) in rice fields using brain acetylcholinesterase (hereafter referred to as AChE) activity as a biomarker. The empirical work, on which this thesis is based, includes structured questionnaires, laboratory and field experiments. First, a field survey using questionnaires was carried out to gain a better understanding of the current state of rice farming systems, the use of pesticides and attitude to pest management strategies among rice and rice-fish farmers, as well as to provide basic information for the set-up of the laboratory and field experiments. Secondly, laboratory studies were conducted to clarify if the selected insecticides applied alone and in mixtures caused negative side effects on climbing perch fingerlings. Thirdly, further toxicity studies were carried out, under rice field conditions, to further investigate the toxicity effects of the insecticides, applied alone, in mixtures and under sequential applications, on climbing perch fingerlings. The results showed that although there were a more selective use of pesticides and an increased awareness among farmers of the negative side effects of pesticides in 2007 as compared to 1999, the current use of pesticide in the Mekong Delta still cause many problems to the environment and human health. Chlorpyrifos ethyl (hereafter referred to as CPF) was found to cause a significant and more prolonged inhibition on the brain AChE activity in climbing perch than fenobucarb (hereafter referred to as F). The inhibition by the mixture of CPF and F were significantly higher tha, At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Submitted. Paper 5: Submitted.

The present study aims to investigate the potential adverse effects of gold nanoparticles (Au NPs) in the cortex, hippocampus, striatum, midbrain, cerebellum and medulla of adult male Wistar rat through the estimation of some oxidative stress parameters and acetylcholinesterase (AChE) activity. Rats were divided into two main experimental groups. Animals of the 1st and 2nd groups were intraperitoneally injected with a single dose (100

The present study aims to investigate the potential adverse effects of gold nanoparticles (Au NPs) in the cortex, hippocampus, striatum, midbrain, cerebellum and medulla of adult male Wistar rat through the estimation of some oxidative stress parameters and acetylcholinesterase (AChE) activity. Rats were divided into two main experimental groups. Animals of the 1st and 2nd groups were intraperitoneally injected with a single dose (100

Flavonoids, a group of natural compounds mainly derived from plants, are known to possess osteogenic effects in bone cells. Here, we aimed to test if flavonoid could induce a cholinergic enzyme, acetylcholinesterase (AChE), as well as bone differentiation. In cultured rat osteoblasts, twenty flavonoids, deriving from Chinese herbs and having known induction of alkaline phosphatase (ALP1) expression, were tested for its induction activity on AChE expression. Eleven flavonoids showed the induction, and five of them had robust activation of AChE expression, including baicalin, calycosin, genistin, hyperin and pratensein: the induction of AChE included the levels of mRNA, protein and enzymatic activity. Moreover, the flavonoid-induced AChE expression in cultured osteoblast was in proline-rich membrane anchor (PRiMA)-linked tetrameric globular form (G4) only. In parallel, the expression of PRiMA was also induced by the application of flavonoids. The flavonoid-induced AChE in the cultures was not affected by estrogen receptor blocker, ICI 182,780. Taken together, the induction of PRiMA-linked AChE in osteoblast should be independent to classical estrogen signaling pathway. © 2016 Elsevier Ireland Ltd

The expression of acetylcholinesterase (AChE), an enzyme hydrolyzes neurotransmitter acetylcholine at vertebrate neuromuscular junction, is regulated during myogenesis, indicating the significance of muscle intrinsic factors in controlling the enzyme expression. DNA methylation is essential for temporal control of myogenic gene expression during myogenesis; however, its role in AChE regulation is not known. The promoter of vertebrate ACHE gene carries highly conserved CG-rich regions, implying its likeliness to be methylated for epigenetic regulation. A DNA methyltransferase inhibitor, 5-azacytidine (5-Aza), was applied onto C2C12 cells throughout the myotube formation. When DNA methylation was inhibited, the promoter activity, transcript expression and enzymatic activity of AChE were markedly increased after day 3 of differentiation, which indicated the putative role of DNA methylation. By bisulfite pyrosequencing, the overall methylation rate was found to peak at day 3 during C2C12 cell differentiation; a SP1 site located at -1826 bp upstream of mouse ACHE gene was revealed to be heavily methylated. The involvement of transcriptional factor SP1 in epigenetic regulation of AChE was illustrated here: (i) the SP1-driven transcriptional activity was increased in 5-Aza-treated C2C12 culture; (ii) the binding of SP1 onto the SP1 site of ACHE gene was fully blocked by the DNA methylation; and (iii) the sequence flanking SP1 sites of ACHE gene was precipitated by chromatin immuno-precipitation assay. The findings suggested the role of DNA methylation on AChE transcriptional regulation and provided insight in elucidating the DNA methylation-mediated regulatory mechanism on AChE expression during muscle differentiation. © 2016 Elsevier B.V. All rights reserved.

Adenosine 5′-triphosphate (ATP), a neurotransmitter and a neuromodulator, has been shown to be co-stored and co-released with acetylcholine (ACh) at the pre-synaptic vesicles in vertebrate neuromuscular junction (nmj). Several lines of studies demonstrated that binding of ATP to its corresponding P2Y1 receptors (P2Y1R) in muscle and neuron regulated the post-synaptic gene expressions. Indeed, the expression of acetylcholinesterase (AChE) in muscle was markedly decreased in P2Y1R−/− (P2Y1R knock-out) mice. In order to search for possible role of P2Y1R in cholinergic function of the brain, the expression of globular form AChE was determined in the brain of P2Y1R−/− mice. In contrast to that in muscle, the amounts of AChE activity, AChE catalytic subunit, structure subunit PRiMA and the amount of ACh, in the brain were not, significantly, altered, suggesting the role of P2Y1R in neuron could have different function as that in muscle. However, the expressions of a series of neuronal development markers, i.e. neurofilaments, were reduced in P2Y1R−/− mouse brain, indicating P2Y1R may be involved in neuronal development process.