Acetylcholinesterase Research Articles

Occurrence of a population of the root-knot nematode, Meloidogyne incognita, with low sensitivity to two major nematicides, fosthiazate and fluopyram, in Japan.

The root-knot nematode (RKN), Meloidogyne incognita, affects food production globally and nematicides, such as fosthiazate and fluopyram, are frequently used in Japan to control damage caused by RKN. In aboveground pests, the emergence of a population with developed resistance is frequently found after the continuous use of the same pesticides; however, there are few studies on changes in the sensitivity of plant-parasitic nematodes, including RKN, to nematicides. We compared the sensitivity of two populations of M. incognita to fosthiazate and fluopyram, one population with a history of exposure to fosthiazate and 1,3-dichloropropene (Ibaraki population) and the other without nematicide use for decades (Aichi population). A concentration of fosthiazate and fluopyram causing 50% mortality at 24 h post-treatment (LC50) was markedly higher in the Ibaraki population (5.4 and 2.3 mg L-1) than in the Aichi population (0.024 and 0.011 mg L-1 in fosthiazate and fluopyram, respectively), indicating the low sensitivity of the Ibaraki population to fosthiazate and fluopyram. Experiments using different enzyme inhibitors indicated the involvement of acetylcholinesterase (AChE), which is the target of fosthiazate, and glutathione S-transferase (GST), a typical enzyme related to detoxification, in the low sensitivity mechanism. The activity of AChE was 33-fold higher in the Ibaraki population than in the Aichi population and there were many differences in their nucleotide sequences. In addition, the gene expression level of GST was 239-fold higher in the Ibaraki population than in the Aichi population. These results revealed differences in the sensitivity to nematicides among RKN populations. Two factors were identified as related to the mechanism of low sensitivity in the Ibaraki population. This is the first report showing the difference in the sensitivity to fluopyram between populations of M. incognita. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Evaluation of <i>Saussurea lappa</i> on Oxidative Stress and Cognition in Aluminium-induced Alzheimer’s Disease Rats

Background: Oxidative stress and neurodegenerative illnesses, such as Alzheimer's disease (AD), are closely associated. There has been a lot of thought put into finding medicinal plants with nootropic properties to slow the onset and course of AD. Objective: The study aimed to evaluate the methanolic extract of Saussurea lappa clarke (MESC) on oxidative stress and cognitive ability induced by aluminium exposure. Methods: Wistar albino rats were chosen for the study. About 30 animals were selected and grouped into 5 with 6 animals in each group. Group I served as control, group II served as disease induced (Aluminium-induced), group III, IV and V were administered with standard drug – Donepezil Hcl, and MESC at two doses – 200 and 400 mg/kg. The behavioural studies were examined by using certain apparatus like Passive Avoidance (PA) test, Elevated Plus Maze, Y- Maze and Actophotometer. Determination of anti-oxidant enzymes – Catalase (CAT) and thiobarbituric acid reactive substances (TBARS) along with acetylcholinesterase (AChE) levels which was done in rat’s brain homogenate. Results: In the PA test, administration of MESC at doses of 200 and 400 mg/kg significantly (**p< 0.01) lengthened step-through latency (STL) in rats on day 30 compared to the positive control group. Animals at MESC (200 & 400 mg/kg) showed noticeably higher memory retention (MR) rates as compared to the disease-control group. Additionally, administration of MESC (200 and 400 mg/kg) significantly (**p< 0.01) raised CAT and declined the concentration of TBARS. AChE concentration was significantly (**p< 0.01) reduced at the dose of MESC at 200 and 400 mg/kg as compared to the positive control group. Conclusion: The present study showed that MESC had a strong nootropic effect on brain antioxidant indicators and cognitive function in rats exposed to aluminium-induced oxidative stress and cognitive impairment. These findings may be investigated in the treatment of neurodegenerative diseases, including AD.

Berberine and palmatine, acting as allosteric potential ligands of α7 nAChR, synergistically regulate inflammation and phagocytosis of microglial cells.

Berberine and palmatine are isoquinoline quaternary alkaloids derived from Chinese medicinal herbs. These alkaloids have shown promising synergy in inhibiting acetylcholinesterase (AChE), indicating their potential in treating Alzheimer's disease (AD). Besides, the anti-inflammatory effects of berberine and palmatine have been widely reported, although the underlying mechanism remains unclear. Here, we found that berberine and palmatine could induce calcium ion (Ca2+) influx via activating α7 nicotinic acetylcholine receptor (α7 nAChR) in cultured microglial cells, possibly serving as its allosteric potential ligands. Furthermore, we examined the synergistic anti-inflammatory effects of berberine and palmatine in the LPS-induced microglia, that significantly suppressed the production of TNF-α and iNOS. Notably, this suppression was reversed by co-treatment with a selective antagonist of α7 nAChR. Moreover, the alkaloid-induced microglial phagocytosis was shown to be mediated by the induction of Ca2+ influx through α7 nAChR and subsequent CaMKII-Rac1-dependent pathway. Additionally, the combination of berberine and palmatine, at low concentration, protected against the LPS-induced endoplasmic reticulum stress and mitochondrial dysfunction in microglia. These findings indicate the potential of berberine and palmatine, either individually or in combination, in contributing to anti-AD drug development, which provide valuable insights into the mechanisms by which natural products, such as plant alkaloids, exert their anti-AD effects.

Rhoifolin, baicalein 5,6-dimethyl ether and agathisflavone prevent amnesia induced in scopolamine zebrafish (Danio rerio) model by increasing the mRNA expression of bdnf, npy, egr-1, nfr2α, and creb1 genes

The increasing attention towards age-related diseases has generated significant interest in the concept of cognitive dysfunction associated with Alzheimer's disease (AD). Certain limitations are associated with the current therapies, and flavonoids have been reported to exhibit multiple biological activities and anti-AD effects in several AD models owing to their antioxidative, anti-inflammatory, and anti-amyloidogenic properties. In this study, we performed an initial in silico predictions of the pharmacokinetic properties of three flavonoids (rhoifolin, baicalein 5,6-dimethyl ether and agathisflavone). Subsequently, we evaluated the antiamnesic and antioxidant potential of flavonoids in concentrations of 1, 3, and 5 μg/L in scopolamine (100 μM)-induced amnesic zebrafish (Danio rerio) model. Zebrafish behavior was analyzed by novel tank diving test (NTT), Y-maze, and novel object recognition test (NOR). Acetylcholinesterase (AChE) activity, brain antioxidant status and the expression of bdnf, npy, egr1, nrf2α, creb1 genes, and CREB-1 protein level was measured to elucidate the underlying mechanism of action. Our flavonoids improved memory and decreased anxiety-like behavior of scopolamine-induced amnesia in zebrafish. Also, the studied flavonoids reduced AChE activity and brain oxidative stress and upregulated the gene expression, collectively contributing to neuroprotective properties. The results of our study add new perspectives on the properties of flavonoids to regulate the evolution of neurodegenerative diseases, especially AD, by modulating the expression of genes involved in the regulation of synaptic plasticity, axonal growth, and guidance, sympathetic and vagal transmission, the antioxidant response and cell proliferation and growth.

9271 Cholinergic Signaling in the Mouse Embryonic Pancreas Targets Early Endocrine Cells

Abstract Disclosure: C. Duarte: None. J.K. Panzer: None. N. Borowsky: None. A. Caicedo: None. The parasympathetic vagus nerve affects pancreas function by activating local intrapancreatic neurons that release acetylcholine (ACh). These neurons are derived from progenitors of the vagal neural crest and colonize the pancreas during embryonic development. The specific mechanisms by which ACh is released from these neurons influence embryonic development of the islet and, specifically, beta cells remain largely unknown. The purpose of this study was to identify cholinergic signaling components and assess early physiological responses to ACh during murine development of endocrine lineage cells. We extracted pancreatic rudiments from mice at embryonic stages E15 to E18 and stained them for neuronal and endocrine markers, along with components associated with cholinergic signaling. We further performed Ca2+ imaging of the pancreatic rudiments from NGN3CRE-GCaMP3 mice to record responses to ACh in cells of the endocrine lineage. At E15, we observed a significantly higher concentration of glucagon+ cells compared to insulin+ cells, consistent with the earlier differentiation of alpha cells in the developing pancreas. Neuronal body clusters were identified in close proximity to endocrine lineage cells from E15 to E18, with the neuronal network becoming more dispersed and interconnected. At E15, wide distribution of the presynaptic vesicular acetylcholine transporter (VAChT) was observed later becoming localized around insulin+ and glucagon+ cells at E18. Postsynaptic acetylcholinesterase (AChE) colocalized with glucagon+ cells from E15 to E18 and with insulin+ cells by E18. ACh elicited Ca2+ responses in individual endocrine cells as early as E15. Our results show that cholinergic nerve terminals target early endocrine cells and elicit responses to ACh, indicating that local cholinergic neurons innervate and activate endocrine progenitors in the embryonic pancreas. Our current studies are aimed at determining how cholinergic signaling contributes to beta cell differentiation and function. We expect this knowledge to guide future protocols for deriving beta cells from stem cells. Presentation: 6/2/2024

8715 Cholinergic Signaling in the Mouse Embryonic Pancreas Targets Early Endocrine Cells

Abstract Disclosure: C. Duarte: None. J.K. Panzer: None. N. Borowsky: None. A. Caicedo: None. The parasympathetic vagus nerve affects pancreas function by activating local intrapancreatic neurons that release acetylcholine (ACh). These neurons are derived from progenitors of the vagal neural crest and colonize the pancreas during embryonic development. The specific mechanisms by which ACh is released from these neurons influence embryonic development of the islet and, specifically, beta cells remain largely unknown. The purpose of this study was to identify cholinergic signaling components and assess early physiological responses to ACh during murine development of endocrine lineage cells. We extracted pancreatic rudiments from mice at embryonic stages E15 to E18 and stained them for neuronal and endocrine markers, along with components associated with cholinergic signaling. We further performed Ca2+ imaging of the pancreatic rudiments from NGN3CRE-GCaMP3 mice to record responses to ACh in cells of the endocrine lineage. At E15, we observed a significantly higher concentration of glucagon+ cells compared to insulin+ cells, consistent with the earlier differentiation of alpha cells in the developing pancreas. Neuronal body clusters were identified in close proximity to endocrine lineage cells from E15 to E18, with the neuronal network becoming more dispersed and interconnected. At E15, wide distribution of the presynaptic vesicular acetylcholine transporter (VAChT) was observed later becoming localized around insulin+ and glucagon+ cells at E18. Postsynaptic acetylcholinesterase (AChE) colocalized with glucagon+ cells from E15 to E18 and with insulin+ cells by E18. ACh elicited Ca2+ responses in individual endocrine cells as early as E15. Our results show that cholinergic nerve terminals target early endocrine cells and elicit responses to ACh, indicating that local cholinergic neurons innervate and activate endocrine progenitors in the embryonic pancreas. Our current studies are aimed at determining how cholinergic signaling contributes to beta cell differentiation and function. We expect this knowledge to guide future protocols for deriving beta cells from stem cells. Presentation: 6/2/2024

GC/MS and LC-ESI-MS Analysis of Conocarpus erectus Leaves Extract via Regulating Amyloid-β-Peptide, Tau Protein, Neurotransmitters, Inflammation and Oxidative Stress against AlCl3-Induced Alzheimer's Disease in Rats.

This study investigated the therapeutic effect of Conocarpus erectus leaves methanolic extract against AlCl3 -induced Alzheimer's disease (AD) in rats comparing with Donepezil-hydrochloride as a reference drug. The bioactive compounds of C. erectus leaves were isolated and identified by GC/MS and LC-ESI-MS analysis. Malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), amyloid-β-peptide (Aβ-peptide), tau protein, acetylcholinesterase (AChE), serotonin (5-HT), dopamine (DA) and nor-adrenaline (NE) levels were estimated. The neuromuscular strength, memory behavior and histopathological examination of cerebral cortex region were also conducted. Forty-three compounds were characterized from the non-polar fraction of C. erectus L. leaves extract and nineteen compounds were identified from the defatted extract. AlCl3- induction caused significant elevation of brain oxidative stress, Aβ-peptide, tau protein, IL-6, TNF-α and AChE levels. A significant decrease in 5-HT, ND and DA levels were noticed. Additionally, AlCl3 reduced neuromuscular strength and compromised memory function. Treatment of AlCl3- induced rats with C. erectuse extract ameliorated these selected parameters by variable degrees. In conclusion, C. erectus protects against AlCl3- induced AD in rats through its antioxidant, anti-inflammatory, and antineutron damage. It could be considered as a new nutraceutical agent for attenuating symptoms associated with Alzheimer's disease.

Assessment of the Interaction of Acetylcholinesterase Binding with Bioactive Compounds from Coffee and Coffee Fractions Digested In Vitro in the Gastrointestinal Tract.

The aim of the study was to evaluate the degree of acetylcholinesterase (AChE) inhibition by green and light- and dark-roasted coffee extracts and their fractions after digestion in a simulated gastrointestinal tract. The analysis was carried out using isothermal titration calorimetry, molecular docking, and dynamics simulations. The results showed that 3-O-caffeoylquinic acid binds strongly to AChE through hydrogen interactions with the amino acids ARG289A, HIS440A, and PHE288A and hydrophobic interactions with TYR121A in the active site of the enzyme. The Robusta green coffee extract (ΔG = -35.87 kJ/mol) and dichlorogenic acid fraction (ΔG = -19-29 kJ/mol) showed the highest affinity. Dichlorogenic acids (3,4-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, and 3,4-O-dicaffeoylquinic acid) have high affinity for AChE as single compounds (ΔG(ITC) = -48.99-55.36 kJ/mol, ΔG(LF/AD) = -43.38-45.38 kJ/mol). The concentration necessary to reduce AChE activity by 50% amounted to 0.22 μmol/μmol chlorogenic acids to the enzyme.

Biogenic synthesis of AgNPs via polyherbal formulation: Mechanistic neutralization and toxicological impact on acetylcholinesterase from Bungarus sindanus venom.

This study aims to examine the biogenic production, characterization, and anti-acetylcholinesterase (AAChE) properties of polyherbal formulation PHF-extract-synthesized silver nanoparticles (PHF-AgNPs). The Elapidae snake Bungarus sindanus has extremely dangerous venom for humans and contains a high amount of AChE (acetylcholinesterase). Inhibiting AChE leads to acetylcholine buildup, affecting neurotransmission. The study tested silver nanoparticles as AChE inhibitors using kinetics. Their production was confirmed through ultraviolet (UV) spectrometry at 425 nm (SPR peak of 1.94), and stabilizing functional groups were identified via Fourier transform infrared spectroscopy (FT-IR). The average length of 20 nm was confirmed by analyzing the scanning electron microscopy (SEM) data. Energy-dispersive X-ray spectroscopy (EDX) identified silver as the primary component of PHF-AgNPs (26%). Statistical analysis showed that the activity of AChE in krait venom decreased by up to 45% and 37% at a given dose of ACh (0.5 mM) by PHF and AgNPs, respectively. Utilizing the Lineweaver-Burk plot for kinetic analysis, a competitive type of inhibition is found. RESEARCH HIGHLIGHTS: Successfully synthesized PHF-extract-induced silver nanoparticles (PHF-AgNPs) demonstrated through UV spectrometry and characterized as crystalline with an average size of 45 nm by X-ray diffraction. PHF-AgNPs effectively inhibited acetylcholinesterase (AChE), an enzyme critical in neurotransmission, reducing its activity in krait venom by up to 45% at certain concentrations. Kinetic analysis revealed that the inhibition mechanism of AChE by PHF-AgNPs is competitive, offering potential for therapeutic applications in neurologically related conditions.

In vitro and Silico Exploration of the Insecticidal Properties of Lavandula multifida L. Essential Oil and its Binary Combinations with Cyantraniliprole and Emamectin benzoate on Spodoptera frugiperda (Lepidoptera: Noctuidae)

Control methods have faced challenges in effectively managing the spread and damage caused by the fall armyworm (FAW), Spodoptera frugiperda, across various crops worldwide, highlighting the need for more sustainable and integrated approaches. This study explored the insecticidal effect of the essential oil (EO) of lavender (LV) (Lavandula multifida L.), cyantraniliprole (CY), and emamectin benzoate (EM) insecticides on FAW larvae. Toxicity, biological, and biochemical assays were employed to elucidate the impact of the two insecticides individually or combined with LV EO. Additionally, in silico molecular docking investigations were performed for eucalyptol (the major component of LV EO) and for CY against the acetylcholine esterase (AChE) enzyme (PDB ID: 6xyu) and cytochrome P450 (PDB ID: 2q6n). EM was the most toxic compound to FAW larvae with an LC50 of 0.0073 mg L−1, followed by CY and LV EO. The chi-square (χ2) test confirmed the synergistic effect of the LV EO/CY combination and the antagonistic effect LV EO/EM combination on FAW larvae. Biological development studies revealed that both LV EO/CY and LV EO/EM combinations impacted the larval and pupal durations. The biochemical assays indicated that the P450-specific activity was activated by LV EO (at LC25), CY (at LC25 & LC50), and their binary combination (at LC25:LC25). In addition, the LV EO/CY combination significantly activated the AChE and GST-specific activity, and the LV EO/CY combination and CY (at LC50) significantly activated α-esterase- specific activity. Molecular docking investigations revealed energy scores (S) of −6.7729 and −7.7644 kcal/mol for eucalyptol and CY, compared to −7.0993 kcal/mol for PDB ID: 6xyu ligand. The current results can serve as baseline data for utilizing the LV EO/CY combination in the integrated pest management (IPM) of FAW.

Toxic Effects of 4-Bromodiphenyl Ether (BDE-3) on Antioxidant Enzymes, Cell Viability, Histology and Biomolecules in Zebrafish Embryo-Larvae.

Polybrominated diphenyl ethers (PBDEs) are a class of flame retardants that are being used widely in industrial and consumers products such as plastics, electronics, furniture, textiles and so forth. They can undergo debromination process to form less brominated diphenyl ethers, which are bioaccumulative, more volatile and more toxic in nature. The current study was conducted to reveal the biochemical, apoptotic, histopathological, ultrastructural and biomolecular (ATR-FTIR) toxicity of 4-bromodiphenyl ether (BDE-3) in zebrafish larvae. After the 96-h LC50 determination, the zebrafish embryos were exposed to sublethal concentrations of BDE-3, that is, 0.79 and 1.59 mg/L. The MDA content was found to be significantly increased in BDE-3 exposed larvae whereas the FRAP activity was found to be decreased. The catalase (CAT), glutathione-S-transferase (GST) and acetylcholinesterase (AChE) activity were observed to be significantly increased, and a decreased superoxide dismutase (SOD) activity was reported after the BDE-3 exposure in zebrafish larvae. The cell viability was reported to be decreased in zebrafish larvae after BDE-3 exposure. Histopathological and ultrastructural alterations were also observed in the BDE-3 exposed zebrafish larvae. The changes in the biomolecules such as DNA and protein were also revealed via ATR-FTIR analysis. The present investigation will help to understand the toxic nature of less brominated diphenyl ethers and could be utilised to assess environmental risk.

Assessment of Neurotoxic Mechanisms of Individual and Binary Mixtures of Cobalt, Nickel and Lead in Hippocampal Neuronal Cells.

Many studies have focused on the neurotoxic effects of single metals, while investigation on the exposure to metal mixtures, which mainly occur in real-life situations, is scarce. This study sought to assess the neurotoxic effect of Ni, Co, and Pb binary mixtures and their individual effects in hippocampal neuronal cells (HT-22). Cells were exposed to Ni, Co, and Pb separately for 48 h at 37°C and 5% CO2, and cell viability was assessed. Morphological assessment of the cells exposed to binary mixtures of Co, Ni, and Pb and single metals was assessed using a microscope. Furthermore, acetylcholinesterase (AChE) activity, oxidative stress biomarkers (glutathione [GSH] and malondialdehyde [MDA] levels, catalase [CAT], and glutathione-S transferase [GST] activities) and nitric oxide [NO] levels were evaluated after treatment with the binary mixtures and single metals. Binary mixtures of the metals reduced cell viability, exerting an additivity action. The combinations also exerted synergistic action, as revealed by the combination index. Furthermore, a significant reduction in AChE activity, GSH levels, CAT and GST activities, and high MDA and NO levels were observed in neuronal cells. The additive interactions and synergistic actions of the binary mixtures might contribute to the significant reduction of AChE activity, GSH levels, GST, and CAT activities, and an increase in MDA and NO levels. The findings from this study revealed significant evidence that binary mixtures of Co, Pb, and Ni may induce impaired neuronal function and, ultimately, neurodegeneration.

Investigating in vitro anticholinergic potential (anti-AChE and anti-BuChE) of Chaenomeles leaves extracts and its phytochemicals including chlorophylls, carotenoids and minerals.

The goal of this work was to evaluate the chemical constitution and health-promoting potential of 12 varieties of Chaenomeles × superba, speciosa and japonica leaves. Carotenoids, chlorophylls, triterpenes, sugars, polyols and acids were analyzed quantitatively and qualitatively using high pressure liquid chromatography (LC) coupled with mass spectrometry (MS), while the mineral profile was determined using atomic absorption spectroscopy (AAS). Moreover, the in vitro anticholinergic potential (inhibition of acetyl-cholinesterase (AChE) and butyryl-cholinesterase (BuChE)) and antioxidant (ABTS, FRAP, ORAC) capacity were evaluated. For the first time in Chaenomeles genotypes 26 carotenoid derivatives and 22 chlorophyll derivatives were identified. Some varieties contained high amounts of carotenoids and chlorophylls (Ch. × superba 'Colour Trail', 'Nicoline', 'Pink Lady', 'Texas Scarlet'), and triterpenes (Ch. speciosa 'Simonii', 'Rubra', and Ch. × superba 'Colour Trail', 'Nicoline') and showed high ORAC antioxidant (Ch. × superba 'Pink Lady' and Ch. speciosa 'Simonii') and anticholinergic (Ch. speciosa species) activity. The studied leaves also contained sugars (3.1 to 16.5mg/100g), organic acids (3.9-8.1g/100g), and minerals (Ca, Cu, Fe, K, Mg, Mn, Na, and Zn). In conclusion, Chaenomeles leaves show potential as a new source for the production of nutraceuticals, as well as for medical and/or cosmetic purposes.

High-sensitive detection of organophosphate and carbamate pesticide residues in milk based on bioluminescence method

Organophosphorus pesticides (OPs) and carbamate pesticides (CBs) are the most commonly used pesticides in agricultural cultivation. The pesticide residues in plant products can easily enter dairy cows through feed, resulting in the pesticide low-concentration residues in milk. Traditional acetylcholinesterase (AChE) inhibition-based colorimetric methods have low sensitivity and could not satisfy the detection of low-concentrations of OPs and CBs residues in dairy products. In this study, we combined firefly luciferase (FLuc)-mediated bioluminescence with the inhibition of AChE-catalyzed substrate activity by pesticides to develop a highly sensitive and rapid method for detecting OPs and CBs residues in milk. AChE breaks down D-luciferin acetate to produce D-luciferin, which is recognized by the FLuc and emits luminescence in the presence of ATP. However, the presence of OPs and CBs inhibits AChE, causing the reduction or disappearance of the luminescence signal. The luminescence signal can be detected using a hand-held luminescence photometer, eliminating the need for large instrumentation. The AChE-FLuc system accurately detected OPs and CBs in milk within 30 min, with detection limits of 7.89 ng/mL and 1.75 ng/mL, respectively. The sensitivity of this method is approximately ten times higher than that of traditional AChE inhibition methods, meeting the pesticide residue limits in milk set by China and the European Union.

A Portable Electrochemical Biosensor Based on an Amino-Modified Ionic Metal-Organic Framework for the One-Site Detection of Multiple Organophosphorus Pesticides.

Constructing stable, portable sensors and revealing their mechanisms is challenging. Ion metal-organic frameworks (IMOFs) are poised to serve as highly effective electrochemical sensors for detecting organophosphorus pesticides (OPs), leveraging their unique charge properties. In this work, an amino-modified IMOF was constructed and combined with near-field communication (NFC) technology to develop a portable, touchless, and battery-free electrochemical biosensor NH2-IMOF@CS@AChE. -NH2 in NH2-IMOF gives the framework a higher electropositivity compared to IMOF, enhancing the electrostatic attraction with acetylcholinesterase (AChE), which is beneficial for immobilizing AChE. Furthermore, the uncoordinated O atoms and the (CH3)2NH2+ groups in NH2-IMOF help to form stronger bonds with AChE through hydrogen bonds. The results showed a wide linear response range of 1 × 10-15 to 1 × 10-9 M and a low detection limit of 1.24 × 10-13 M for glyphosate (Gly) in the practical detection of OPs. Additionally, electrochemical biosensor arrays were constructed to effectively identify and distinguish multiple OPs on the basis of their unique differential pulse voltammetry (DPV) electrochemical signals. This work provides a simple and effective solution for on-site OP analysis and can be widely applied in food safety and water quality monitoring.

Alkaloids from Stephania cephalantha and their enzyme inhibitory activities

A new protoberberine type alkaloid, 11-diethylaminomethyl corydalmine (1), and 27 known alkaloids (2-28) were isolated from the tuberous roots of Stephania cephalantha. Their structures were identified by spectral analysis. Enzyme inhibitory activities of the compounds on acetylcholinesterase (AChE), tyrosinase and α-glucosidase were evaluated. Compounds 13, 16 and 18 exhibited comparable AChE inhibitory activities to the positive control huperzine A, with the IC50 values of 0.046, 0.048, and 0.057 mg/mL, respectively.

A-088 Clinically Significant Errors Using the Diazyme Total Bile Acid Assay on the Roche c502 Analyzer: Investigating a Result Error Identifies a Novel Large-Scale Testing Error Caused by Reagent Carryover

Abstract Background Thoroughly investigating single patient result errors may identify systemic, large-scale testing errors. The laboratory observed an event where a revision to a Total Bile Acid (TBA, Diazyme Laboratories, Inc.) patient result following a 22S quality control (QC) failure displayed a larger than expected difference (52 to 5mcmol/L). TBA is primarily used for diagnosis and monitoring of intrahepatic cholestasis of pregnancy with results >10mcmol/L considered elevated. Retrospective review of result revisions following QC failures (3/15/2017-5/30/2023) revealed that 50%(20/40) yielded result differences of >10mcmol/L(>20SD based on assay imprecision). The aim of this study was to apply a systematic approach to i) estimate the rate of TBA errors, ii) ensure accurate result reporting during investigation period, iii) perform root cause analysis (RCA), and iv) determine corrective and preventative action(s). Methods Residual samples from TBA testing (6/24-7/5/2023) were retested(n=158), differences >+/-3.0mcmol/L or 15% were confirmed on an alternate analyzer, and reports were revised. Automated repeat testing of TBA samples >10mcmol/L was operationalized 7/6/2023. Initial and repeat TBA results were compared(n=448) and results differing by >+/-20% were remeasured on an alternate analyzer. RCA was conducted using a fishbone diagram. NaOH reagent probe washes were implemented for TBA and the error rate was re-assessed. Assays run prior to TBA samples with errors (n=15) were identified. To assess reagent carryover a residual serum pool (TBA ∼5mcmol/L) was aliquoted into 5 tubes in a sample rack. The first sample in the rack was programmed to run an assay suspected of causing carryover followed by 4 TBA measurements. Mean±standard deviation(SD) TBA concentrations were calculated. TBA was also measured in the liquid reagent for amylase, lipase, acetylcholinesterase (ACE) and fructosamine. Results Initial TBA retesting yielded no errors when initial TBA was ≤10mcmol/L(n=51). For samples with TBA >10mcmol/L(n=107), 9(8.4%) had differences exceeding criteria with 8/9 being revised to ≤10mcmol/L. Analysis of automated patient repeat data showed a 3.8%(17/448) error rate when initial TBA >10mcmol/L. After NaOH reagent probe washes were implemented, the error rate decreased to 0.7%(3/448). Assays run directly before an erroneously high TBA result included: lactate, fructosamine, soluble transferrin receptor(STFR), lipase, and ACE. A serum TBA pool(mean±SD=5.1±0.4 mcmol/L,n=15) measured 37.6±1.2mcmol/L(n=3) and 6.7±0.7mcmol/L(n=3) after lipase and fructosamine, respectively. No other assays demonstrated carryover. TBA in lipase reagent compartment B and C was 653 and 649mcmol/L, respectively, and 653mcmol/L in fructosamine reagent compartment B. Conclusions A single patient TBA result revision was investigated and led to identification of reagent carryover causing erroneously high TBA results on the Roche c502 analyzer. Automatic, real-time, repeat testing was implemented to prevent reporting incorrect patient results until RCA could identify the cause. Occurrence rate of erroneous high TBA results decreased from 3.8% to 0.7% after implementation of NaOH washes on the instruments. Carryover experiments confirmed that lipase and fructosamine assays cause carryover when run prior to a TBA sample due to measurable TBA in the reagent. The lab is pursuing moving the fructosamine and lipase reagent to the Roche Cobas c701 to prevent the issue.

Fe3O4@MXene@PEI aerogel immobilized acetylcholinesterase for inhibitor screening from herbal plants

Screening of acetylcholinesterase (AChE) inhibitors is a common strategy in drug discovery for treating Alzheimer’s disease. Herein, AChE was immobilized onto magnetic polyethyleneimine-based MXene aerogels through both electrostatic interactions and covalent bonds, enabling its application in AChE activity assays and inhibitor screening of herbal plants. The composite was analyzed using a range of techniques, including scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), and Zeta potential analysis, to gain insight into its chemical and physical properties. The proposed Fe3O4@MXene@PEI-AChE composite exhibited enhanced temperature and pH stability, as evaluated by Ellman’s method, along with good reusability. The Michaelis Menten constant (Km) of the immobilized AChE was calculated to be 0.68 mmol/L. Additionally, an inhibition kinetic study was conducted to verify the feasibility of utilizing the immobilized enzyme to screen for inhibitors, with huperzine A employed as a model inhibitor. The proposed strategy was employed to compare the AChE inhibitory activity of 18 commonly used herbal medicines for treating AD, revealing both the aqueous and alcoholic extracts of Coptis chinensis as exhibiting the highest AChE inhibitory activity. Finally, the screening of AChE inhibitors in Coptis chinensis extracts were conducted by combining the proposed strategy with UPLC-Q-Orbitrap high resolution mass spectrometry. This study presents a feasible strategy for monitoring AChE activity and holds considerable potential for further exploration of AChE-inhibiting active ingredients in herbal medicines.

Holistic exploration of silver nanoparticles synthesized from Carthamus oxycantha leaf extracts: Characterization and assessment of antioxidant, anti-α-amylase, and anti-cholinesterase activities using comprehensive statistical methods

In the burgeoning field of nanotechnology, plant-mediated nanoparticles (NPs) have attracted significant attention due to their small size, favorable chemical properties, and eco-friendly nature. We utilized Carthamus oxycantha leaf extract to synthesize silver nanoparticles (AgNPs) and evaluate their potential as antioxidants and inhibitors of acetylcholinesterase (AChE) and alpha-amylase (α-amylase). The AgNPs were characterized using advanced techniques. The UV–Vis spectrum recorded at 410 nm, with an absorbance of 2.3 a.u., confirmed the presence of AgNPs. Fourier transform infrared spectroscopy (FT-IR) indicated that functional groups from the extract were attached to the AgNP surface, aiding in their formation and stabilization. X-ray diffraction analysis (XRD) revealed four major peaks at 37, 43, 64, and 77, confirming AgNP synthesis. Scanning electron microscopy (SEM) showed that 80 % of the AgNPs were spherical, measuring 25–50 nm, while energy dispersive X-ray (EDX) analysis indicated that silver constituted 83 % of the material. The AgNPs demonstrated significant scavenging activity: 59.3 % with ABTS, 67.2 % with ammonium molybdate, 60.35 % with DPPH, 65.2 % with ferric chloride, and 55.2 % with H2O2. Both α-amylase and AChE were inhibited by AgNPs, exhibiting uncompetitive inhibition characteristics. For glucophage, Km decreased from 143 to 75 µg, and Vmax decreased from 45 % to 72 % as concentrations increased from 50 to 150 µg. The Km of AgNPs decreased from 25 to 17 µg, and Vmax decreased from 16 % to 37 %. The extract showed noncompetitive inhibition against α-amylase, while AgNPs caused noncompetitive inhibition in AChE. These findings highlight the potential of AgNPs as therapeutic agents for managing oxidative stress and enzyme-related disorders, paving the way for future research and applications in medicinal chemistry.

Apocynaceae as a Potential Source for Acetylcholinesterase Inhibition in Symptomatic Regulation and Management of Alzheimer's Disease

: Memory loss or dementia is the key symptom of Alzheimer's disease (AD). In AD, significant interference in a progressive manner is observed in memory, behaviour, and cognitive abilities that affect the daily life of a person. At present, more than 50 million people are affected worldwide with Alzheimer's disease. Urgent attention is needed for the symptomatic regulation and management of this disease. The significant pharmacotherapy research in the last two decades gave only four drug compounds galanthamine, donepezil, rivastigmine, and memantine that inhibit the enzyme acetylcholinesterase (AChE) to elevate the availability of acetylcholine in the brain for symptomatic relief in AD patients. Plant-based AChE inhibitors from many plant families, mainly including Rutaceae, Papaveraceae, Apocynaceae, Rubiaceae, Amaryllidaceae, Liliaceae, Lycopodiaceae, Fabaceae, Lamiaceae, etc., have been characterized for the management of AD progression. AD progression is described by cholinergic, amyloid, Tau protein, oxidative stress, and neuroinflammatory hypothesis. To date, there is no comprehensive review in the literature that combined all plants of the Apocynaceae family showing anti-AChE activity. Therefore, the current review aims to present significant literature, especially on plant-derived compounds from the Apocynaceae family that inhibit AChE. The review compiled all plants showing potent anti-acetylcholinesterase activity. The anti-AChE activity of more than 30 plants is described, which may be potential targets to find new drug molecules by attracting the attention of researchers toward the Apocynaceae family. More than 8 species of genus Tabernaemontana of Apocynaceae have been investigated for indole alkaloids, demonstrating AChE inhibitory activity. The majority of anti-AChE compounds belong to the class of alkaloids.