The Potential Organ Involved in Cantharidin Biosynthesis in Epicauta chinensis Laporte (Coleoptera: Meloidae).

Cantharidin, a terpenoid defensive toxin mainly produced by blister beetles, is among the most widely known insect natural products in the world. However, little is known about the site of cantharidin biosynthesis in vivo. Our previous research showed that 3-hydroxy-3-methylglutary-CoA reductase (HMGR) is an essential enzyme in cantharidin biosynthesis. In this report, we further investigated cantharidin titer and HMGR mRNA expression levels in different tissues of male and female Epicauta chinensis, and performed a comparative analysis of HMGR transcript levels in male Tenebrio molitor, a Tenebrionidae beetle that cannot produce cantharidin. HMGR transcripts had a positive correlation with cantharidin production. Furthermore, the specifically high amounts of HMGR transcript and abundant cantharidin production in fat body of male E. chinensis indicated the process of cantharidin synthesis may occur in the fat body.

PSMB4 expression associates with epithelial ovarian cancer growth and poor prognosis.

PURPOSE: In this study, we investigated the expression and role of PSMB4 in human epithelial ovarian cancer(EOC). METHODS: Western blot was used to evaluate the expression of PSMB4 in EOC tissues, and immunohistochemical analysis was performed on 115 cases of ovarian cancers. Then, we used Fisher exact test to analyze the correlation between PSMB4 and clinicopathological parameters. Starvation and re-feeding assay was used to assess cell cycle. CCK-8 assay and plate colony formation assay showed the influence of PSMB4 on proliferation of EOC cells. RESULTS: The expression of PSMB4 in EOC tissues was higher than normal ovary tissues and was significantly associated with clinical pathologic variables. Kaplan-Meier curve showed that high expression of PSMB4 was related to poor prognosis of EOC patients. Starvation and re-feeding assay suggested that PSMB4 played a critical role in EOC cell proliferation. CCK-8 assay and plate colony formation assay showed that EOC cells treated with PSMB4-siRNA reduced cell proliferation of EOC cells. Additionally, PSMB4 knockdown decreased NF-κB activity. PSMB4 also regulated the expression of NF-κB mediated proteins, including cyclin D1, and cyclin E which involved in cell proliferation. CONCLUSIONS: Our findings implied that PSMB4 is involved in the progression of EOC and could serve as potential therapeutical target of EOC. These data suggested that PSMB4 may promote cell proliferation via the NF-κB-target gene in EOC.

Annexin A2 plays a critical role in epithelial ovarian cancer.

PURPOSE: This study aimed at investigating the potential role and prognostic significance of Annexin A2 in human epithelial ovarian cancer (EOC). METHODS: Western blot was used to evaluate the expression of Annexin A2 in nine fresh EOC tissues, and immunohistochemical analysis was performed on formalin-fixed paraffin-embedded sections of 119 cases of ovarian cancers. Then, we used Fisher exact test to analyze the correlation between Annexin A2 and clinicopathological parameters. Starvation refeeding was used to detect the alteration of Annexin A2 in HO8910 cell cycle. RESULTS: Annexin A2 was overexpressed in carcinoma tissues compared with normal tissue, and the expression levels gradually increased from G1 to G3. Moreover, the staining of tissue microarray was consistent with the result we got from western blot, increasing from G1 to G3 gradually, and it was related to the Figo stage (P = 0.005), histologic grade (P = 0.002), ascite (P < 0.001), malignant tumor cells (P < 0.001), residual tumor size (P = 0.044), Ki-67 (P = 0.003). Kaplan-Meier analysis revealed that high Annexin A2 expression was significantly associated with poor prognoses of the patients (P < 0.001). Multivariate analysis demonstrated that Annexin A2 was an independent prognostic indicator for overall survival. Starvation refeeding indicated that Annexin A2 was related to EOC cell proliferation. CONCLUSIONS: We could hypothesize that Annexin A2 acted a critical role in EOC cell proliferation, and may be used as a potential and novel therapeutic target for EOC. These data suggest that Annexin A2 may promote the progression of EOC and be a therapeutic target for EOC therapy.

Expression of the non-neuronal cholinergic system components in Malpighian tubules of Mythimna separata and evidence for non-neuronal acetylcholine synthesis.

The non-neuronal cholinergic system, widely distributed in nature, is an ancient system that has not been well studied in insects. This study aims to investigate the key components of the cholinergic system and to identify the non-neuronal acetylcholine (ACh)-producing cells and the acting sites of ACh in the Malpighian tubules (MTs) of Mythimna separata. We found that non-neuronal ACh in MTs is synthesized by carnitine acetyltransferase (CarAT), rather than choline acetyltransferase (ChAT), as confirmed by using enzyme inhibitors and high-performance liquid chromatography coupled with mass spectrometry (HPLC-MS/MS). Fluorescence in situ hybridization revealed the presence of CarAT mRNA within MTs, specifically localized in the principal cells. Immunohistochemistry showed strong staining for A-mAChR, a muscarinic acetylcholine receptor, in the principal cells. Pharmacological analysis further demonstrated that ACh acts through A-mAChR in the principal cells to increase the intracellular Ca2+ concentration. These findings provide compelling evidence for the existence of a non-neuronal cholinergic system in the MTs of M. separata, and the principal cells play a crucial role in ACh synthesis via CarAT.

Arsenic sulfide enhances radiosensitivity in rhabdomyosarcoma via activating NFATc3-RAG1 mediated DNA double strand break (DSB).

Rhabdomyosarcoma (RMS) represents one of the most lethal soft-tissue sarcomas in children. The toxic trace element arsenic has been reported to function as a radiosensitizer in sarcomas. To investigate the role of arsenic sulfide (As4S4) in enhancing radiation sensitization in RMS, this study was conducted to elucidate its underlying mechanism in radiotherapy. The combination of As4S4 and radiotherapy showed significant inhibition in RMS cells, as demonstrated by the cell counting kit-8 (CCK-8) assay and flow cytometry. Subsequently, we demonstrated for the first time that As4S4, as well as the knockdown of NFATc3 led to double-strand break (DSB) through increased expression of RAG1. In vivo experiment confirmed that co-treatment efficiently inhibited RMS growth. Furthermore, survival analysis of a clinical cohort consisting of 59 patients revealed a correlation between NFATc3 and RAG1 expression and overall survival (OS). Cox regression analysis also confirmed the independent prognostic significance of NFATc3 and RAG1.Taken together, As4S4 enhances radiosensitivity in RMS via activating NFATc3-RAG1 mediated DSB. NFATc3 and RAG1 are potential therapeutic targets. As4S4 will hopefully serve as a prospective radio-sensitizing agent for RMS.

Identification and biochemical characterization of protein phosphatase 5 from the cantharidin-producing blister beetle, Epicauta chinensis.

Protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5) was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat) motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5) was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate) and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.

SDO-Based Command Filtered Adaptive Neural Tracking Control for MIMO Nonlinear Systems With Time-Varying Constraints.

In this article, an adaptive neural tracking control based on saturation disturbance observer (SDO) and command filter is studied for multiple-input-multiple-output nonlinear systems with time-varying constraints and system uncertainties. By employing neural networks (NNs), the system uncertainties are approximated. The SDO is proposed to estimate the composited disturbances which consist of NN approximation errors and the external bounded disturbances. Compared with the traditional disturbance observer, the SDO can reduce the estimation error to some extent. The control requirements are achieved based on the multiconstraints which contain three layers: 1) prescribed performance functions (PPFs); 2) actual constraints; and 3) virtual constraints. The errors remain within the prescribed small neighborhood of zero by using the PPFs, the error constraints ensure that the time-varying constraints are never violated even if the PPFs are not available, and the virtual constraints are applied in a new time-varying barrier Lyapunov function (TVBLF) to design virtual controllers and controller to solve the singularity problem of the traditional TVBLF. In addition, the command filter is introduced to solve the problem of "explosion of complexity." Finally, a numerical simulation verifies the effectiveness of the proposed scheme for a flight control of unmanned aerial vehicle.

Adaptive NN Tracking Control for Uncertain MIMO Nonlinear System With Time-Varying State Constraints and Disturbances.

In this article, an adaptive neural network (NN) tracking control scheme is proposed for uncertain multi-input-multi-output (MIMO) nonlinear system in strict-feedback form subject to system uncertainties, time-varying state constraints, and bounded disturbances. The radial basis function NNs (RBFNNs) are adopted to approximate the system uncertainties. By constructing the intermediate variables, the external disturbances that cannot be directly measured are approximated by the disturbance observers. The time-varying barrier Lyapunov function (TVBLF) is constructed to guarantee the boundedness of the errors lie in the sets. To overcome the potential singularity problem that the denominator of the barrier function term approaches zero in controller design, the adaptive NN tracking control scheme with time-varying state constraints is proposed. Based on the TVBLF, the controller will be designed to guarantee tracking performance without violating the appropriate error constraints. The analysis of TVBLF shows that all closed-loop signals remain semiglobally uniformly ultimately bounded (SGUUB). The simulation results are performed to validate the validity of the proposed scheme.

Arsenic Sulfide Inhibits Hepatocellular Carcinoma Metastasis by Suppressing the HIF-1α/VEGF Pathway.

BACKGROUND: Metastasis is one of the principal reasons of cancer mortality from hepatocellular carcinoma (HCC). The goal of our investigation was to examine the mechanism by which arsenic sulfide (As4S4) represses the metastasis of HCC. METHODS: The cell counting kit-8 (CCK-8) assay was conducted to observe cell viability of HCC cell lines HepG2 and Hep3B following As4S4 treatment, and their metastasis was studied using the wound-healing and transwell assays. HCC-induced angiogenesis of human umbilical vein endothelial cells (HUVEC) was assessed by tube formation assay. Enzyme-linked immunosorbent assay (ELISA), western blot, quantitative polymerase chain reaction and immunofluorescence staining were utilized to evaluate key molecules involved in metastasis, including hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), Vimentin, N-cadherin and E-cadherin. RESULTS: As4S4 suppressed the proliferation, migration and invasion of HepG2 and Hep3B cell lines in a concentration-dependent pattern, and inhibited HCC cell-induced angiogenesis of HUVEC in the tube formation assay. Treatment with As4S4 also decreased the expression of crucial elements involved in the metastasis of HCC cells, including HIF-1α and VEGF, while reducing epithelial-mesenchymal transition, as shown by Western blot, ELISA and immunofluorescence staining. CONCLUSIONS: Overall, results above indicate that As4S4 suppresses the metastasis of HCC cells via the HIF-1α/VEGF pathway.

Regioselective Hydroxylation of Flavonoids by Transition-Metal-Catalyzed C-H Bond Oxidation.

Regioselective synthesis of 5,6,7-trihydroxyl and 5,7,8-trihydroxyl flavones has been achieved via a transition-metal-catalyzed C-H oxidation as the key step using naturally enriched 5,7-dihydroxyl flavone. The developed chemistry was applied to the synthesis of the naturally occurring and biologically active flavonoids wogonin (2), oroxylin A (3), and their glycosylated derivatives (4 and 5) as potential carnitine palmitoyltransferase 1 activators.

Construction of a fatty acid metabolism-related gene signature for predicting prognosis and immune response in breast cancer.

Background: Breast cancer has the highest incidence among malignant tumors in women, and its prevalence ranks first in global cancer morbidity. Aim: This study aimed to explore the feasibility of a prognostic model for patients with breast cancer based on the differential expression of genes related to fatty acid metabolism. Methods: The mRNA expression matrix of breast cancer and paracancer tissues was downloaded from The Cancer Genome Atlas database. The differentially expressed genes related to fatty acid metabolism were screened in R language. The TRRUST database was used to predict transcriptional regulators related to hub genes and construct an mRNA-transcription factor interaction network. A consensus clustering approach was used to identify different fatty acid regulatory patterns. In combination with patient survival data, Lasso and multivariate Cox proportional risk regression models were used to establish polygenic prognostic models based on fatty acid metabolism. The median risk score was used to categorize patients into high- and low-risk groups. Kaplan-Meier survival curves were used to analyze the survival differences between both groups. The Cox regression analysis included risk score and clinicopathological factors to determine whether risk score was an independent risk factor. Models based on genes associated with fatty acid metabolism were evaluated using receiver operating characteristic curves. A comparison was made between risk score levels and the fatty acid metabolism-associated genes in different subtypes of breast cancer. The differential gene sets of the Kyoto Encyclopedia of Genes and Genomes for screening high- and low-risk populations were compared using a gene set enrichment analysis. Furthermore, we utilized CIBERSORT to examine the abundance of immune cells in breast cancer in different clustering models. Results: High expression levels of ALDH1A1 and UBE2L6 prevented breast cancer, whereas high RDH16 expression levels increased its risk. Our comprehensive assessment of the association between prognostic risk scoring models and tumor microenvironment characteristics showed significant differences in the abundance of various immune cells between high- and low-risk breast cancer patients. Conclusions: By assessing fatty acid metabolism patterns, we gained a better understanding of the infiltration characteristics of the tumor microenvironment. Our findings are valuable for prognosis prediction and treatment of patients with breast cancer based on their clinicopathological characteristics.

Event-Triggered Fractional-Order Tracking Control for an Uncertain Nonlinear System With Output Saturation and Disturbances.

In this article, an event-triggered (ET) fractional-order adaptive tracking control scheme (ATCS) is studied for the uncertain nonlinear system with the output saturation and the external disturbances by using the nonlinear disturbance observer (NDO) and the neural networks (NNs). Based on NNs, the system uncertainties are approximated. An NN-based NDO is designed to estimate the bounded disturbances. Combining the NNs, the output of the designed NDO, the fractional-order theory, and the ET mechanism, an ATCS is proposed under the output saturation. According to the stability analysis, all the closed-loop signals are semiglobally uniformly ultimately bounded based on the investigative ATCS. The simulation results and the comparative experiment verifications are shown to indicate the viability of the developed control scheme.

Potential key roles of tumour budding: a representative malignant pathological feature of non-small cell lung cancer and a sensitive indicator of prognosis.

OBJECTIVES: To investigate the relationship between tumour budding, clinicopathological characteristics of patients and prognosis in non-small cell lung cancer. STUDY DESIGN: A retrospective study was used. PARTICIPANTS: We selected 532 patients with non-small cell lung cancer from China, including 380 patients with adenocarcinoma and 152 with squamous cell carcinoma. PRIMARY AND SECONDARY OUTCOME MEASURES: Tumour budding was visible using H&E staining as well as pancytokeratin staining. The count data and measurement data were compared using the χ2 test and the t-test, respectively. The overall survival rate was the follow-up result. The survival curves were drawn using the Kaplan-Meier method, and the differences between groups were analysed using the log-rank method. The independent prognostic factor of patients with lung cancer was determined using a multivariate Cox proportional hazard model. RESULTS: In patients with lung adenocarcinoma, there was a correlation between tumour budding and spread through air spaces (OR 36.698; 95% CI 13.925 to 96.715; p<0.001), and in patients with squamous cell carcinoma, tumour budding state was closely related to the peritumoural space (OR 11.667; 95% CI 4.041 to 33.683; p<0.001). On Cox regression analysis, multivariate analysis showed that tumour budding, pleural and vascular invasion, spread through air spaces, tumour size, lymph node metastasis, and tumour node metastasis stage were independent risk factors of prognosis for patients with non-small cell lung cancer. CONCLUSIONS: As an effective and simple pathological diagnostic index, it is necessary to establish an effective grading system in the clinical diagnosis of lung cancer to verify the value of tumour budding as a prognostic indicator. We hope that this analysis of Chinese patients with non-small cell lung cancer can provide useful reference material for the continued study of tumour budding.

Comparative transcriptome analysis reveals the non-neuronal cholinergic system in the ovary of the oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae).

BACKGROUND: Acetylcholine (ACh), as a classical neurotransmitter, plays great roles in the nervous system. There is increasing evidence of its non-neuronal roles in regulating basic cell functions in vertebrates. However, knowledge about the non-neuronal cholinergic system in insects is scarce. RESULTS: A comparative transcriptome analysis was performed to investigate differences in the key molecular components of the cholinergic system between the head and ovary. The results showed that expression levels of most cholinergic system-related genes were higher in the head than in the ovary, and some cholinergic components were absent in the ovary. ACh contents ranged from 0.1 to 1.3 µg mg-1 of wet weight during the development of the ovary, and weak acetylcholinesterase activity was also detected. Moreover, the ovary has a capacity for ACh synthesis. Bromoacetylcarnitine (BrACar), a specific carnitine acetyltransferase (CarAT) inhibitor, greatly inhibits ACh synthesis by 83.83% in ovary homogenates, but bromoacetylcholine (BrACh), a specific choline acetyltransferase (ChAT) inhibitor, has no effect on ACh synthesis in the ovary. These findings indicate that non-neuronal ACh in the ovary is only catalyzed by CarAT. CONCLUSION: This study reveals the existence of the non-neuronal cholinergic system in the ovary of M. separata, whose synthesis and release mechanisms are different from those of the head. These results provide novel insights into the non-neuronal cholinergic system in insects, and will be valuable in the discovery of new target genes and the future development of green pest control. © 2022 Society of Chemical Industry.

A Comprehensive Prognostic and Immune Analysis of Ferroptosis-Related Genes Identifies SLC7A11 as a Novel Prognostic Biomarker in Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is still one of the illnesses with the greatest mortality and morbidity. As a recently identified mode of cellular death, the activation of ferroptosis may promote the effectiveness of antitumor therapies in several types of tumors. However, the expression and clinical significance of Ferroptosis-associated genes in LUAD are still elusive. The RNA sequencing data of LUAD and relevant clinical data were downloaded from The Cancer Genome Atlas (TCGA) datasets. Subsequently, potential prognostic biomarkers were determined by the use of biological information technology. The R software package "ggalluvial" was applied to structure Sanguini diagram. Herein, our team screened 14 dysregulated ferroptosis-associated genes in LUAD. Among them, only four genes were associated with clinical outcome of LUAD patients, including ATP5MC3, FANCD2, GLS2, and SLC7A11. In addition, we found that high SLC7A11 expression predicted an advanced clinical progression in LUAD patients. Additionally, 8 immune checkpoint genes and 7 immune cells for LUAD were recognized to be related to the expression of SLC7A11. KEGG assays indicated that high expression of SLC7A11 might participate in the modulation of intestinal immune network for IgA generation and Staphylococcus aureus infection. Overall, our findings revealed that SLC7A11 might become a potentially diagnostic biomarker and SLC7A11 might serve as an independent prognosis indicator for LUAD.

Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant, Polyrhachis vicina.

Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full-length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599-amino acid protein that exhibits a high degree of homology with other mAchRs. Real-time quantitative RT-PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains.

Draft genome of the blister beetle, Epicauta chinensis.

Existence of cantharidin (CTD) in blister beetles is a significant ecological adaptive mechanism that defends against predators and regulates courtship and mating behaviors. To better understand CTD biosynthetic information as well as its biology and pharmacology, we assembled a genome of 151.88 Mb for Epicauta chinensis using PacBio sequencing technology. Gene annotation yielded 249,238 repeats, 527 non-coding RNAs and 12,520 protein-coding genes. Compared to other 11 insects, expansions of gene families in E. chinensis for most core gene families likely associated with environmental adaptation, such as chemoreception, immunity, and detoxification. We further annotated P450s and immune-related genes, a total of 117 putative P450s comprising 7 CYP2, 67 CYP3, 36 CYP4, and 7 mitochondrial P450s and 281 immune-related genes were identified. Comparative analysis of the insect immune repertoires indicated presence of immune genes detected only from Coleopteran insects such as MD2-like. This suggested a lineage-specific gene evolution for Coleopteran insects. Based on the gene family evolution analysis, we identified two probable candidate genes including CYP4TT1 and phytanoyl-CoA dioxygenase for CTD biosynthesis. The high-quality reference genome of E. chinensis provides the genetic basis for further investigation of CTD biosynthesis and in-depth studies of the development and evolution of blister beetles.

Nanochannel-based sensor for the detection of lead ions in traditional Chinese medicine.

Lead ions (Pb2+) are used in the quality control of traditional Chinese medicine (TCM) preparations because they are highly toxic to human health. At present, sophisticated analytical instrumentation and complicated procedures for sample analysis are needed for the determination of Pb2+. Herein, a simple, fast, and sensitive peptide-modified nanochannel sensor to detect Pb2+ in TCM is reported, which is based on a Pb2+-specific peptide modified porous anodized aluminum membrane (PAAM). This peptide-based nanochannel clearly has the highest selectivity for Pb2+ when compared to other heavy metal ions, including As2+, Cd3+, Co2+, Cr2+, Cu2+, Fe3+, Hg2+, Mg2+, Mn2+, Ni2+, and Zn2+. Based on linear ranges from 0.01 to 0.16 µM and 10 to 100 µM, the detection limit was calculated to be 0.005 µM. Moreover, this peptide-based nanochannel sensor was successfully used to detect Pb2+ in complex TCM samples. In addition, when compared with the gold standard atomic absorption spectrophotometry (AAS) method, the recovery of the peptide-modified nanochannel sensor was between 87.7% and 116.8%. The experimental results prove that this new sensor is able to achieve accurate detection of Pb2+ in TCM samples. Thus, this sensor system could provide a simple assay for sensitive and selective detection of Pb2+ in TCM, thereby showing great potential in the practical application for the quality control of heavy metals in TCM.

Progressive muscle relaxation for patients with chronic schizophrenia: A randomized controlled study.

PURPOSE: To evaluate progressive muscle relaxation (PMR) as an intervention for anxiety, psychotic symptoms, and quality of life (QOL) in patients with chronic schizophrenia. DESIGN AND METHODS: Eighty patients were recruited from a Taiwanese psychiatry ward. The intervention group received group PMR; control group received treatment-as-usual. RESULTS: The results indicated that PMR might have a short-term effect on reducing anxiety, improving psychotic syndromes, and QOL among patients with chronic schizophrenia; however, the effectiveness at the 3-month follow-up was not evident. PRACTICE IMPLICATIONS: Both the psychiatric patients and the health institutions may be able to list PMR as a clinical routine care, and then become a mental health practice strategy for mental patients to improve the quality of mental care. IMPLICATIONS FOR NURSING PRACTICE: Our studies suggest that prevention of severe mental illness among patients with schizophrenia requires PMR interventions. PMR had an immediate effect, and it is possible that a shorter intervention period using this approach would also be successful.

Characterization of an A-Type Muscarinic Acetylcholine Receptor and Its Possible Non-neuronal Role in the Oriental Armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae).

Muscarinic acetylcholine receptor (mAChR) regulates many neurophysiological functions in insects. In this report, a full-length cDNA encoding an A-type mAChR was cloned from the oriental armyworm, Mythimna separata. Pharmacological properties studies revealed that nanomolar to micromolar concentrations of carbachol or muscarine induced an increase of intracellular Ca2+ concentration ([Ca2+] i ), with the EC50 values of 124.6 and 388.1 nM, respectively. The increases of [Ca2+] i can be greatly blocked by the antagonist atropine, with an IC50 value of 0.09 nM. The receptor mRNA is expressed in all developmental stages, with great differential expression between male and female adults. The tissue expression analysis identified novel target tissues for this receptor, including ovaries and Malpighian tubules. The distribution of Ms A-type mAChR protein in the male brain may suggest the neurophysiological roles that are mediated by this receptor. However, the receptor protein was found to be distributed on the membranes of oocytes that are not innervated by neurons at all. These results indicate that Ms A-type mAChR selectively mediates intracellular Ca2+ mobilization. And the high level of receptor protein in the membrane of oocytes may indicate a possible non-neuronal role of A-type mAChR in the reproductive system of M. separata.