Biochemical and molecular assessment of oxidative stress in fruit fly exposed to azo dye Brilliant Black PN.

BACKGROUND: Azo dyes are widely used in the food industry to prevent color loss during processing and storage of products. This study aimed to investigate the effect of a diazo dye Brilliant Black PN (E151) on oxidative stress-related parameters in fruit flies (Drosophila melanogaster) at biochemical and molecular levels. METHODS AND RESULTS: Third instar larvae were transferred to a medium containing the dye at different doses (1, 2.5, and 5 mg/mL). Gene expression and activity of superoxide dismutase, catalase (CAT), glutathione peroxidase (GPX), and acetylcholinesterase (AChE) enzymes were determined in the heads of adult flies obtained from these larvae. In addition, the glutathione (GSH) and malondialdehyde levels were measured using spectrophotometric analysis. Mitochondrial DNA (mtDNA) copy number was also detected by real-time PCR. The results showed that treatment with 5 mg/mL of the dye caused a decrease in both gene expression and enzyme activity of CAT and GPx. Moreover, the same dose of dye treatment decreased AChE activity, GSH level, and mtDNA copy number. CONCLUSIONS: As a result, Brilliant Black PN dye can trigger toxicity by altering the level and activity of oxidative stress-related biomarkers in a dose-dependent manner. Therefore, more comprehensive studies are needed to elucidate the side effect mechanism and toxicity of this dye.

A MnO2 nanosheet-mediated CRISPR/Cas12a system for the detection of organophosphorus pesticides in environmental water.

Nowadays, easy, convenient, and sensitive sensing strategies are still critical for organophosphorus pesticides in environmental water samples. Herein, a novel organophosphorus pesticide (OP) assay based on acetylcholinesterase (AChE) and a MnO2 nanosheet-mediated CRISPR/Cas12a reaction is reported. The single-strand DNA (ssDNA) activator of CRISPR/Cas12a was simply adsorbed on the MnO2 nanosheets as the nanoswitches of the assay. In the absence of target OPs, AChE hydrolyzed acetylcholine (ATCh) to thiocholine (TCh), which reduced the MnO2 nanosheets to Mn2+, resulting in the release of the activator followed by activation of the CRISPR/Cas12a system. The activated Cas12a thereafter nonspecifically cleaved the FAM/BHQ1-labeled ssDNA (FQ-reporter), producing a fluorescence signal. Upon the addition of target OPs, the hydrolysis of ATCh by AChE was inhibited owing to OPs combining with AChE, and thus effective quantification of OPs could be achieved by measuring the fluorescence changes of the system. As a proof of concept, dichlorvos (DDVP) was chosen as a model OP analyte to address the feasibility of the proposed method. Attributed to the excellent trans-cleavage activity of Cas12a, the fluorescent biosensor exhibits a satisfactory limit of detection (LOD) for DDVP at 0.135 ng mL-1. In addition, the excellent recoveries for the detection of DDVP in environmental water samples demonstrate the applicability of the proposed assay in real sample research.

Identification of ACHE as the hub gene targeting solasonine associated with non-small cell lung cancer (NSCLC) using integrated bioinformatics analysis.

Background: Solasonine, as a major biological component of Solanum nigrum L., has demonstrated anticancer effects against several malignancies. However, little is understood regarding its biological target and mechanism in non-small cell lung cancer (NSCLC). Methods: We conducted an analysis on transcriptomic data to identify differentially expressed genes (DEGs), and employed an artificial intelligence (AI) strategy to predict the target protein for solasonine. Subsequently, genetic dependency analysis and molecular docking were performed, with Acetylcholinesterase (ACHE) selected as a pivotal marker for solasonine. We then employed a range of bioinformatic approaches to explore the relationship between ACHE and solasonine. Furthermore, we investigated the impact of solasonine on A549 cells, a human lung cancer cell line. Cell inhibition of A549 cells following solasonine treatment was analyzed using the CCK8 assay. Additionally, we assessed the protein expression of ACHE, as well as markers associated with apoptosis and inflammation, using western blotting. To investigate their functions, we employed a plasmid-based ACHE overexpression system. Finally, we performed dynamics simulations to simulate the interaction mode between solasonine and ACHE. Results: The results of the genetic dependency analysis revealed that ACHE could be identified as the pivotal target with the highest docking affinity. The cell experiments yielded significant findings, as evidenced by the negative regulatory effect of solasonine treatment on tumor cells, as demonstrated by the CCK8 assay. Western blotting analysis revealed that solasonine treatment resulted in the downregulation of the Bcl-2/Bax ratio and upregulation of cleaved caspase-3 protein expression levels. Moreover, we observed that ACHE overexpression promoted the expression of the Bcl-2/Bax ratio and decreased cleaved caspase-3 expression in the OE-ACHE group. Notably, solasonine treatment rescued the Bcl-2/Bax ratio and cleaved caspase-3 expression in OE-ACHE cells compared to OE-ACHE cells without solasonine treatment, suggesting that solasonine induces apoptosis. Besides, solasonine exhibited its anti-inflammatory effects by inhibiting P38 MAPK. This was supported by the decline in protein levels of IL-1ß and TNF-α, as well as the phosphorylated forms of JNK and P38 MAPK. The results from the molecular docking and dynamics simulations further confirmed the potent binding affinity and effective inhibitory action between solasonine and ACHE. Conclusions: The findings of the current investigation show that solasonine exerts its pro-apoptosis and anti-inflammatory effects by suppressing the expression of ACHE.

Transcriptome-based analyses reveal venom diversity in two araneomorph spiders, Psechrus triangulus and Hippasa lycosina.

The spiders Psechrus triangulus and Hippasa lycosina are widely distributed in Yunnan Province, China, and are important natural enemies of agricultural pests, yet studies regarding the composition of their venom are lacking. In this study, cDNA libraries were constructed from venom gland tissue of P. triangulus and H. lycosina and used for transcriptomic analysis. From the analysis, 39 and 31 toxin-like sequences were predicted for P. triangulus and H. lycosina, respectively. The predicted neurotoxin sequences were categorized according to cysteine sequence motifs, and the predicted neurotoxin sequences of P. triangulus and H. lycosina could be classified into 9 and 6 toxin families, respectively. In addition, potential acetylcholinesterase, hyaluronidase, and astaxanthin-like metalloproteinases were identified through annotation. In summary, transcriptomic techniques were invaluable in mining the gene expression information from these two spider species to explore the toxin composition of their venom and determine how they differ. Studies of this type provide essential baseline data for studying the evolution and physiological activities of spider toxins and for the potential development of medicinal compounds.

Serotonin Receptors and Acetylcholinesterase Gene Expression Alternations: The Potential Value on Tumor Microenvironment of Gastric Cancer.

INTRODUCTION: Gastric cancer is one of the common causes of cancer-related death in the world. Neurotransmitters have recently been related to the proliferation of cancer cells, but the role of neurotransmitters in the progression of gastric cancer is still unexplored. The cross-talk between the nervous system and immune cells through serotonin and its receptors in the tumor microenvironment can impact tumor progress. Our purpose is to expose probable changes in serotonin receptors, acetylcholinesterase, and monoamine oxidase A gene expression in gastric cancer. METHODS: Transcript of serotonin receptors (5-HTR2A, 5-HTR2B, 5-HTR3A, 5-HTR7) and monoamine oxidase A genes in the peripheral blood mononuclear cells (40 patients and 40 control) and tissue (21 tumors and 21 normal adjacent tissues) were assessed. The gene expression was analyzed by quantitative real-time PCR using suitable primers. Statistical analysis was performed using appropriate software (REST, Prism). RESULTS: Significantly higher amounts of 5-HTR2A, 5-HTR2B, 5-HTR3A, 5-HTR7, and acetylcholinesterase gene transcripts were found in the peripheral blood of gastric cancer patients compared with healthy individuals. The expression of 5-HTR2B and 5-HTR3A genes was significantly higher (p = 0.0250, p = 0.0005, respectively) and the acetylcholinesterase gene was lower in the tissue of patients (p = 0.0119) compared with adjacent normal tissue. CONCLUSION: This study highlights the role of serotonin receptors in gastric cancer that might have suggestions for the development of novel therapeutics and defensive approaches that target factors associated with the link between the nervous system, cancer cells, and the tumor microenvironment.

Fenpyroximate induced cytotoxicity and genotoxicity in Wistar rat brain and in human neuroblastoma (SH-SY5Y) cells: Involvement of oxidative stress and apoptosis.

Fenpyroximate (FEN) is an acaricide used in agriculture / horticulture to control spider mites and leafhoppers. It inhibits the transport of mitochondrial electrons at the level of NADH-coenzyme Q oxidoreductase (complex I). Despite the implication of inhibition of mitochondrial complex I in neurotoxicity, especially in neurodegenerative diseases, data concerning FEN neurotoxicity remain limited. Thus, the present study was designed to investigate the toxic effect of FEN on rat brain tissue and on human neuroblastoma cells (SH-SY5Y). Rat exposure to FEN at three different doses (4.8, 9.6 and 48 mg / Kg bw) for 28 consecutive days resulted in histopathological modifications in brain tissue and a significant decrease in acetylcholinesterase activity. Further, FEN significantly enhanced lipid peroxidation and protein oxidation in rat brain and disturbed activities of antioxidant enzymes (SOD, CAT, GPx, and GST). Besides, FEN was found to induce DNA damage in a significant and dose-dependent manner in rat brain as assessed by the comet assay. To better understand FEN neurotoxic effect, we monitored our study on SH-SY5Y cells. We confirm our data found in rat brain tissue. In fact, FEN induced cell mortality in a concentration dependent manner. It over-produced intracellular ROS and lipid peroxidation and enhanced SOD and CAT activities. FEN was also found to induce DNA damage in SH-SY5Y cells. Moreover, FEN induced a loss of mitochondrial membrane potential, which confirms FEN mitochondrial impairing activity. Acridine Orange-Bromure Etidium (AO-BE) cell staining indicated that FEN enhanced the percentage of apoptotic cells in a concentration dependent manner. Further, pretreatment with a general caspases inhibitor (ZVAD-FMK), reduced significantly the FEN induced cell mortality. We also shown that FEN increased caspase 3 activity. These findings suggested, for the first time, the possibility of the involvement of mitochondrial pathway in FEN-induced cell apoptosis.

CRISPR-Cas12a based fluorescence assay for organophosphorus pesticides in agricultural products.

Herein, we propose a sensitive fluorescent assay for organophosphorus pesticides (OPs) detection based on a novel strategy of activating the CRISPR-Cas12a system. Specifically, acetylcholinesterase (AChE) hydrolyzes acetylthiocholine into thiocholine (TCh). Subsequently, TCh induces the degradation of MnO2 nanosheets and generates sufficient Mn2+ ions to activate the Mn2+-dependent DNAzyme. Then, as the catalytic product of activated DNAzyme, the short DNA strand activates the CRISPR-Cas12a system to cleave the fluorophore-quencher-labeled DNA reporter (FQ) probe effectively; thus, increasing the fluorescence intensity (FI) in the solution. However, in the presence of OPs, the activity of AChE is suppressed, resulting in a decrease in FI. Under optimized conditions, the limits of detection for paraoxon, dichlorvos, and demeton were 270, 406, and 218 pg/mL, respectively. Benefiting from the outstanding MnO2 nanosheets properties and three rounds of enzymatic signal amplification, the proposed fluorescence assay holds great potential for the detection of OPs in agricultural products.

Insecticide resistance in pepper greenhouse populations of Aphis gossypii (Hemiptera: Aphididae) in Korea.

The cotton aphid or melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), is a polyphagous insect pest with a wide host range. Two distinct genetic clusters were found in A. gossypii populations in Korea. To determine whether the division of the genetic clusters was driven by insecticide selection pressure, the frequencies of insecticide resistance-associated mutations on three representative insecticide target genes [i.e., nicotinic acetylcholine receptor gene (nAChR), voltage-gated sodium channel gene (vgsc), and acetylcholinesterase 1 gene (ace-1)] were predicted in A. gossypii populations with known genetic structures. Most populations revealed heterozygosity-resistant alleles for the nAChR R81T and vgsc M918L mutations, but homozygous-resistant alleles for the ace-1 S431F mutation. However, assessment of the three mutation frequencies revealed no apparent correlation between the genetic structures and the resistance profiles. The regression analysis revealed no correlation between the genetic cluster ratios and resistance allele frequencies (R81T, S431F, and M918L). We used three insecticides that are commonly used in greenhouses: imidacloprid (neonicotinoid), acephate (organophosphate), and esfenvalerate (pyrethroid), to test resistance and susceptibility in A. gossypii populations. The bioassay results revealed that the BS_19 (Busan) and JE_19 (Jeongeup) populations were resistant to imidacloprid and acephate, the HS_19 (Honseong) population was resistant to acephate and esfenvalerate, and susceptible lab strains only exhibited resistance to acephate. The bioassay results were correlated with mutation frequency, but no correlation was detected among genetic clusters. These results suggest that the distinct genetic structure observed in the Korean populations of A. gossypii is not likely influenced by insecticide resistance traits, but rather by other factors.

Senescence and oxidative stress toxicities induced by lamivudine and tenofovir in Drosophila melanogaster.

BACKGROUND: Lamivudine and tenofovir disoproxil fumarate act against the replication of hepatitis B and human immunodeficiency viruses via inhibition of the reverse transcriptase enzyme activity, thereby preventing the synthesis of viral DNA. Chronic administration of these drugs has been associated with toxicities, including senescence, oxidative stress and premature death. A study of these toxicities in Drosophila melanogaster, which share 75% genomic similarity with humans could help to develop a pharmacologic intervention. METHODS: Susceptibility of D. melanogaster for lamivudine and tenofovir-induced toxicities were investigated. First, flies (≤3 days old) were fed with drugs-supplemented diet at varying concentrations (1mg to 300mg/10-gram diet) or distilled water for seven days to determine LD50. Secondly, five groups of 60 flies were fed with four concentrations of test drugs: 2.9mg, 5.82mg, 11.64mg and 23.28mg each per 10-gram diet for 28 days survival and lifespan assays. Then 5-day treatment plan was utilized to determine drugs toxicities on climbing ability and some biomarkers of oxidative stress. Finally, molecular docking was carried out using the Auto-dock vina mode to predict the biological interactions between the test drugs and D. melanogaster acetylcholinesterase (AChE) or glutathione-S-transferase (GST). RESULTS: The LD50 of lamivudine or tenofovir was 47.07 or 43.95mg/10g diet, respectively. Each drug significantly (P<0.05) reduced the survival rate, longevity and climbing performance of the flies dose-dependently. These drugs also altered levels of biochemical parameters: AChE, GST, superoxide dismutase (SOD), catalase (CAT), total thiol (T-SH), and malondialdehyde (MDA) of the flies significantly (P<0.05). In silico molecular analysis showed that the test drugs interacted with significantly (P<0.05) higher binding affinities at the same catalytic sites of D. melanogaster GST and AChE compared with substrates (glutathione or acetylcholine). CONCLUSION: The significant lamivudine and tenofovir-induced toxicities observed as increased mortality, climbing deficits and compromised antioxidant defence in D. melanogaster demands further research for possible pharmacological intervention.

A network pharmacology-based approach to explore mechanism of action of medicinal herbs for alopecia treatment.

Hair loss is one of the most common skin problems experienced by more than half of the world's population. In East Asia, medicinal herbs have been used widely in clinical practice to treat hair loss. Recent studies, including systematic literature reviews, indicate that medicinal herbs may demonstrate potential effects for hair loss treatment. In a previous study, we identified medical herbs used frequently for alopecia treatment. Herein, we explored the potential novel therapeutic mechanisms of 20 vital medicinal herbs for alopecia treatment that could distinguish them from known mechanisms of conventional drugs using network pharmacology analysis methods. We determined the herb-ingredient-target protein networks and ingredient-associated protein (gene)-associated pathway networks and calculated the weighted degree centrality to define the strength of the connections. Data showed that 20 vital medicinal herbs could exert therapeutic effects on alopecia mainly mediated via regulation of various target genes and proteins, including acetylcholinesterase (AChE), phospholipase A2 (PLA2) subtypes, ecto-5-nucleotidase (NTE5), folate receptor (FR), nicotinamide N-methyltransferase (NNMT), and quinolinate phosphoribosyltransferase (QPRT). Findings regarding target genes/proteins and pathways of medicinal herbs associated with alopecia treatment offer insights for further research to better understand the pathogenesis and therapeutic mechanism of medicinal herbs for alopecia treatment with traditional herbal medicine.

Regulation of acetylcholinesterase during the lipopolysaccharide-induced inflammatory responses in microglial cells.

The non-classical function of acetylcholine (ACh) has been reported in neuroinflammation that represents the modulating factor in immune responses via activation of α7 nicotinic acetylcholine receptor (α7 nAChR), i.e., a cholinergic anti-inflammatory pathway (CAP). Acetylcholinesterase (AChE), an enzyme for ACh hydrolysis, has been proposed to have a non-classical function in immune cells. However, the involvement of AChE in neuroinflammation is unclear. Here, cultured BV2 cell, a microglial cell line, and primary microglia from rats were treated with lipopolysaccharide (LPS) to induce inflammation and to explore the regulation of AChE during this process. The expression profiles of AChE, α7 nAChR, and choline acetyltransferase (ChAT) were revealed in BV2 cells. The expression of AChE (G4 form) was induced significantly in LPS-treated BV2 cells: the induction was triggered by NF-κB and cAMP signaling. Moreover, ACh or α7 nAChR agonist suppressed the LPS-induced production of pro-inflammatory cytokines, as well as the phagocytosis of microglia, by activating α7 nAChR and followed by the regulation of NF-κB and CREB signaling. The ACh-induced suppression of inflammation was abolished in AChE overexpressed cells, but did not show a significant change in AChE mutant (enzymatic activity knockout) transfected cells. These results indicate that the neuroinflammation-regulated function of AChE may be mediated by controlling the ACh level in the brain system.

Influence of GSTM1, T1 genes polymorphisms on oxidative stress and liver enzymes in rural and urban pesticides-exposed workers.

Several studies discussed the relationship between the toxicity of organophosphates (OPs) and carbamates pesticides and oxidative stress which affects human health. This study aimed to evaluate the effects of pesticides on the induction of oxidative stress and hepatotoxicity. It was also focused on glutathione-S-transferase gene polymorphism in the modulation of these effects. In addition, the role of the educational level of exposed workers was studied. Acetylcholinesterase (AChE), butyrylcholinesterase (BuChE), liver enzymes, malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and glutathione S transferase (GST) were estimated at 100 pesticide-exposed workers (50 urban researchers (UE) and 50 rural sprayers (RE)), and 100 matched controls (50 urban controls (UC)and 50 rural controls (RC)). AChE and BuChE were decreased in RE and UE compared to RC. Aspartate aminotransferase (AST) and alkaline phosphatase (ALP) activity were elevated in UE and UC compared to the RE and RC. Alanine aminotransferase (ALT) was elevated in UE compared to RE. MDA in RE and UE showed elevation compared to RC. There was a significant reduction in the levels of GSH, GST, and GPx in UE compared to RE and RC. The most sensitive pesticide-induced hepatotoxicity group were exposed workers with the GSTT1 genotype. Within these workers, ALT and ALP were significantly correlated with MDA and inversely correlated with AChE and BuChE, while AST was inversely correlated with AChE and BuChE only in UE. Conclusion: GST gene polymorphisms appeared to have a significant role in workers' susceptibility to hepatotoxic effects due to occupational exposure to pesticides; GSTT1 was the most sensitive genotype.

Multifarious Functions of Butyrylcholinesterase in Neuroblastoma: Impact of BCHE Deletion on the Neuroblastoma Growth In Vitro and In Vivo.

The physiological functions of butyrylcholinesterase (BChE) and its role in malignancy remain unexplained. Our studies in children newly diagnosed with neuroblastoma indicated that BChE expressions is proportional to MYCN amplification suggesting that pathogenesis of high-risk disease may be related to the persistent expression of abnormally high levels of tumor-associated BChE. BChE-deficient neuroblastoma cells (KO [knockout]) were produced from MYCN -amplified BE(2)-C cells (WT [wild-type]) by the CRISPR-Cas9 targeted disruption of the BCHE locus. KO cells have no detectable BChE activity. The compensatory acetylcholinesterase activity was not detected. The average population doubling time of KO cells is 47.0±2.4 hours, >2× longer than WT cells. Reduced proliferation rates of KO cells were accompanied by the loss of N-Myc protein and a significant deactivation of tyrosine kinase receptors associated with the aggressive neuroblastoma phenotype including Ros1, TrkB, and Ltk. Tumorigenicity of WT and KO cells in male mice was essentially identical. In contrast, KO xenografts in female mice were very small (0.37±0.10 g), ~3× smaller compared with WT xenografts (1.11±0.30 g). Unexpectedly, KO xenografts produced changes in plasma BChE similarly to WT tumors but lesser in magnitude. The disruption of BCHE locus in MYCN -amplified neuroblastoma cells decelerates proliferation and produces neuroblastoma cells that are less aggressive in female mice.

Geographical distribution and origin of acetylcholinesterase mutations conferring acaricide resistance in Tetranychus urticae populations from Turkey.

Two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae), is a cosmopolitan pest species that can feed on more than 1000 host plant species. Historically, organophosphate (OP) and carbamate insecticides have been used to control this extremely polyphagous pest. However, its ability to develop acaricide resistance rapidly has led to failure in control. Mutations in acetylcholinesterase gene (ace), the target-site of OP and carbamate insecticides, have been reported to be one of the major mechanisms underlying this developing resistance. In this study, mutations previously associated with resistance (G119S, A201S, T280A, G328A, F331W/Y) in ace have been screened in 37 T. urticae populations collected across Turkey. All mutations were found in various populations, except G119S. Almost all populations had F331W/Y mutation (being fixed in 32 populations), whereas only two populations harboured A201S mutation, but not fixed. On the other hand, more than half of the populations contained T280A and G328A mutations. In addition, the presence of same haplotypes in populations originating from distinct geographic locations and a wide variety of ace haplotypes might indicate multiple origins of F331W and F331Y mutations; however, this needs further investigation. The results of area-wide screening showed that ace mutations are widely distributed among T. urticae populations. Therefore, the use of this group of insecticides should be limited or only rotational use might be regarded as a resistance management tool due to its different mode of action from other main acaricide groups in T. urticae control across Turkey.

Acetylcholinesterase and human cancers.

The enzyme acetylcholinesterase (AChE) is a serine hydrolase whose primary function is to degrade acetylcholine (ACh) and terminate neurotransmission. Apart from its role in synaptic transmission, AChE has several "non-classical" functions in non-neuronal cells. AChE is involved in cellular growth, apoptosis, drug resistance pathways, response to stress signals and inflammation. The observation that the functional activity of AChE is altered in human tumors (relative to adjacent matched normal tissue) has raised several intriguing questions about its role in the pathophysiology of human cancers. Published reports show that AChE is a vital regulator of oncogenic signaling pathways involving proliferation, differentiation, cell-cell adhesion, migration, invasion and metastasis of primary tumors. The objective of this book chapter is to provide a comprehensive overview of the contributions of the AChE-signaling pathway in the growth of progression of human cancers. The AChE isoforms, AChE-T, AChE-R and AChE-S are robustly expressed in human cancer cell lines as well in human tumors (isolated from patients). Traditionally, AChE-modulators have been used in the clinic for treatment of neurodegenerative disorders. Emerging studies reveal that these drugs could be repurposed for the treatment of human cancers. The discovery of potent, selective AChE ligands will provide new knowledge about AChE-regulatory pathways in human cancers and foster the hope of novel therapies for this disease.

Neuroprotective effects of NDEELNK from sea cucumber ovum against scopolamine-induced PC12 cell damage through enhancing energy metabolism and upregulation of the PKA/BDNF/NGF signaling pathway.

The aim of the study was to evaluate the neuroprotective function of sea cucumber ovum peptide-derived NDEELNK and explore the underlying molecular mechanisms. NDEELNK exerted the neuroprotective effect by improving the acetylcholine (ACh) level and reducing the acetylcholinesterase (AChE) activity in PC12 cells. By molecular docking, we confirmed that the NDEELNK backbone and AChE interacted through hydrophobic and hydrogen bonds in contact with the amino acid residues of the cavity wall. NDEELNK increased superoxide dismutase (SOD) activity and decreased reactive oxygen species (ROS) production, thereby reducing mitochondrial dysfunction and enhancing energy metabolism. Our results demonstrated that NDEELNK supplementation alleviated scopolamine-induced PC12 cell damage by improving the cholinergic system, increasing energy metabolism and upregulating the expression of phosphorylated protein kinase A (p-PKA), brain-derived neurotrophic factor (BNDF) and nerve growth factor (NGF) signaling proteins in in vitro experiments. These results demonstrated that the sea cucumber ovum peptide-derived NDEELNK might play a protective role in PC12 cells.

Chlorpyrifos induces cell proliferation in MCF-7 and MDA-MB-231 cells, through cholinergic and Wnt/ß-catenin signaling disruption, AChE-R upregulation and oxidative stress generation after single and repeated treatment.

Chlorpyrifos (CPF) biocide, is associated with breast cancer. The processes underlying this association have not been elucidated to date. CPF increases MCF-7 and MDA-MB-231 cell proliferation after acute and long-term treatment, partially through KIAA1363 overexpression and aryl-hydrocarbon receptor activation but also through estrogen receptor-alpha activation after 24 h exposure in MCF-7 cells, suggesting other mechanisms may be involved. CPF induces reactive oxygen species (ROS) generation, acetylcholine accumulation, and overexpression of acetylcholinesterase-R/S (AChE-R/S) variants, while it also alters the Wnt/ß-catenin pathway, both in vitro and in vivo, in processes different from cancer. These latter mechanisms are also linked to cell proliferation and could mediate this effect induced by CPF. Our results show that CPF (0.01-100 µM), following one-day and fourteen-days treatment, respectively, induced ROS generation and lipid peroxidation, and acetylcholine accumulation due to AChE inhibition, Wnt/ß-catenin up- or downregulation depending on the CPF treatment concentration, and AChE-R and AChE-S overexpression, with the latter being mediated through GSK-3ß activity alteration. Finally, CPF promoted cell division through ACh and ROS accumulation, AChE-R overexpression, and Wnt/ß-catenin signaling disruption. Our results provide novel information on the effect of CPF on human breast cancer cell lines that may help to explain its involvement in breast cancer.

Expression of Drosophila melanogaster acetylcholinesterase (DmAChE) gene splice variants in Pichia pastoris and evaluation of its sensitivity to organophosphorus pesticides.

Acetylcholinesterase (AChE) is a key enzyme used to detect organophosphorus pesticide residues by the enzyme inhibition method. An accidental discovery of a mutant strain with AChE activity was made in our laboratory during the process of AChE expression by Pichia pastoris. The pPIC9K-Drosophilamelanogaster acetylcholinesterase (DmAChE)-like expression vector was constructed by codon optimization of this mutant strain, which was transformed into P. pastoris GS115, and positive clones were selected on yeast peptone dextrose (YPD) plate with G418 at 4.0 mg/mL. The GS115-pPIC9K-DmAChE-like strain was subjected to 0.5% methanol induction expression for 120 h, with a protein band at 4.3 kDa found by the tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) pattern of the fermentation supernatant. After preliminary purification by ammonium sulfate precipitation, the enzyme activity was detected to be 76.9 U/(mL⋅min). In addition, the pesticide sensitivity test proved that DmAChE-like is selective and sensitive to organophosphorus pesticides.

Modulatory effects of caffeic acid on purinergic and cholinergic systems and oxi-inflammatory parameters of streptozotocin-induced diabetic rats.

Diabetes mellitus (DM) is a metabolic disorder characterized by a chronic hyperglycemia state, increased oxidative stress parameters, and inflammatory processes. AIMS: To evaluate the effect of caffeic acid (CA) on ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and adenosine deaminase (ADA) enzymatic activity and expression of the A2A receptor of the purinergic system, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzymatic activity and expression of the α7nAChR receptor of the cholinergic system as well as inflammatory and oxidative parameters in diabetic rats. METHODS: Diabetes was induced by a single dose intraperitoneally of streptozotocin (STZ, 55 mg/kg). Animals were divided into six groups (n = 10): control/oil; control/CA 10 mg/kg; control/CA 50 mg/kg; diabetic/oil; diabetic/CA 10 mg/kg; and diabetic/CA 50 mg/kg treated for thirty days by gavage. RESULTS: CA treatment reduced ATP and ADP hydrolysis (lymphocytes) and ATP levels (serum), and reversed the increase in ADA and AChE (lymphocytes), BuChE (serum), and myeloperoxidase (MPO, plasma) activities in diabetic rats. CA treatment did not attenuate the increase in IL-1ß and IL-6 gene expression (lymphocytes) in the diabetic state; however, it increased IL-10 and A2A gene expression, regardless of the animals' condition (healthy or diabetic), and α7nAChR gene expression. Additionally, CA attenuated the increase in oxidative stress markers and reversed the decrease in antioxidant parameters of diabetic animals. CONCLUSION: Overall, our findings indicated that CA treatment positively modulated purinergic and cholinergic enzyme activities and receptor expression, and improved oxi-inflammatory parameters, thus suggesting that this phenolic acid could improve redox homeostasis dysregulation and purinergic and cholinergic signaling in the diabetic state.

Preparation of a Bombyx mori acetylcholinesterase enzyme reagent through chaperone protein disulfide isomerase co-expression strategy in Pichia pastoris for detection of pesticides.

The cholinesterase-based spectrophotometric methods for detection of organophosphate pesticides (OPs) and carbamate pesticides (CPs) have been proposed as a good choice for their high efficiency, simplicity and low cost. The enzyme, as a core reagent, is of great importance for the developed method. In this study, a protein disulfide isomerase (PDI) co-expression strategy in Pichia pastoris was employed to enhance the yield of recombinant Bombyx mori acetylcholinesterase 2 (rBmAChE2). Subsequently, the prepared enzyme reagent was used to detect the pesticides in real samples. The results showed that the co-expression of rBmAChE2 with PDI increased the enzyme activity of the supernatant and the yield of purified rBmAChE2 up to 60 U/mL and 6 mg/L respectively, both almost 5-fold higher than those of original recombinant strain. In addition, 5 g/L gelatin reagent could help to preserve nearly 90% of the rBmAChE2 activity for 90 days in 4°C and the limits of detections (LODs) of the rBmAChE2-based assay for 20 kinds of OPs or CPs ranged from 0.010 to 2.725 mg/kg, which were lower than most of indexes present in current Chinese National Standard (GB/T 5009.199-2003) or the maximum residue limits (GB 2763-2019). Furthermore, the detection results of 23 vegetable samples were verified by the ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method, which indicated that the rBmAChE2-based assay in this work is suitable for pesticide residues rapid detection.