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Temperature-responsive yeast cell-imprinted sensors (CIPs/AuNPs/Ti 3 C 2 T x /AuNPs/Au) were prepared based on fluorescein isothiocyanate labeled yeast cells (FITC-yeast) via metal-free visible-light-induced atom transfer radical polymerization (MVL ATRP). Here, N -isopropyl acrylamide (NIPAM) was used as a temperature-responsive functional monomer, α-methacrylic acid (MAA) was chosen as an auxiliary functional monomer, N , N '-methylene bisacrylamide (MBA) was used as a cross-linker, and FITC-yeast was selected as both a template and photocatalyst. Under the optimal conditions, the detection range of the yeast cell-imprinted sensor toward yeast cells was 1.0 × 10 2 to 1.0 × 10 9 cells per mL, and the detection limit was 11 cells per mL (S/N = 3), with a linear equation of Δ I (μA) = 8.44 log[ C (cells per mL)] + 7.62 ( R 2 = 0.993). The sensor showed good selective recognition in the presence of interfering substances such as autolyzed yeast cells (AY), dead yeast cells (DY), human mammary epithelial cells (MCF-10A), human breast cancer cells (MCF-7) and Escherichia coli (EC). The sensor also had good consistency and reproducibility. Finally, spiked recovery experiments were performed to investigate the recognition of yeast cells in the actual sample using the yeast cell-imprinted sensor. The spiked recoveries were all in the range of 98.5-108.0%, and the RSD values were all less than 4%, indicating that the sensor had good application prospects.

Background: Insect cytochrome P450 monooxygenases (P450s) play a key role in the detoxification metabolism of insecticides and their overexpression is often associated with insecticide resistance. Our previous research showed that the overexpression of four P450 genes is responsible for clothianidin resistance in B. odoriphaga. In this study, we characterized another P450 gene, CYP6FV21, associated with clothianidin resistance. However, the molecular basis for the overexpression of P450 genes in clothianidin-resistant strain remains obscure in B. odoriphaga., Results: In this study, the CYP6FV21 gene was significantly overexpressed in the clothianidin-resistant (CL-R) strain. Clothianidin exposure significantly increased the expression level of CYP6FV21. Knockdown of CYP6FV21 significantly increased the susceptibility of B. odoriphaga larvae to clothianidin. The transcription factor Cap 'n' Collar isoform-C (CncC) was highly expressed in the midgut of larvae in B. odoriphaga. The expression level of CncC was higher in the CL-R strain compared with the susceptible (SS) strain. Clothianidin exposure caused reactive oxygen species (ROS) accumulation and significantly increased the expression level of CncC. Knockdown of CncC caused a significant decrease in the expression of CYP3828A1 and CYP6FV21, and P450 enzyme activity, and led to a significant increase in mortality after exposure to lethal concentration at 30% (LC 30 ) of clothianidin. After treatment with CncC agonist curcumin, the P450 activity and the expression levels of CYP3828A1 and CYP6FV21 significantly increased, and larval sensitivity to clothianidin decreased. The ROS scavenger N-acetylcysteine (NAC) treatment significantly inhibited the expression levels of CncC, CYP3828A1 and CYP6FV21 in response to clothianidin exposure and increased larval sensitivity to clothianidin., Conclusion: Taken together, these results indicate that activation of the CncC pathway by the ROS burst plays a critical role in clothianidin resistance by regulating the expression of CYP3828A1 and CYP6FV21 genes in B. odoriphaga. This study provides more insight into the mechanisms underlying B. odoriphaga larval resistance to clothianidin. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

Cytochrome P450 monooxygenases (P450s) play important roles in the detoxification metabolism of xenobiotics and are involved in the resistance of insects to many insecticides. In this study, piperonyl butoxide (PBO), an inhibitor of P450 enzyme activity, significantly increased the toxicity of clothianidin in the clothianidin-resistant (CL-R) population of Bradysia odoriphaga . The enzyme activity of P450 in the CL-R population was significantly higher than that in the SS population. Furthermore, four P450 genes were found to be significantly overexpressed in the CL-R population. Tissue-specific expression analysis indicates that CYP9J57 , CYP3828A1 , CYP6SX1 , and CYP6QE1 were most highly expressed in the midgut and/or Malpighian tubules. After exposure to LC 30 of clothianidin, the expression levels of the four P450 genes were significantly upregulated. The RNAi-mediated knockdown of CYP9J57 , CYP3828A1 , and CYP6QE1 significantly increased the susceptibility of B. odoriphaga to clothianidin. These results suggest that P450 genes are involved in clothianidin resistance in B. odoriphaga . This provides a better understanding of P450-mediated clothianidin resistance in B. odoriphaga and will contribute to the management of insect resistance to insecticides.

Carboxylesterases (CarEs) are a multigene superfamily of metabolic enzymes involved in metabolic detoxification of xenobiotics. In this study, an α - esterase gene ( BoαE1 ) was identified from Bradysia odoriphaga . Phylogenetic analysis classified BoαE1 into the α- esterase clade. Developmental expression analysis indicated that BoαE1 was significantly expressed in the second to fourth larval stages. Tissue-specific expression analysis indicated that BoαE1 was highly expressed in the larval midgut. After exposure to LC 30 of malathion, the CarE activity of B. odoriphaga was induced and the transcriptional level of BoαE1 was significantly up-regulated. Silencing of BoαE1 significantly increased the susceptibility of B. odoriphaga larvae to malathion. Inhibition assays in vitro indicated that malathion significantly inhibited BoαE1 activity. GC-MS assay showed that BoαE1 possesses hydrolase activity toward malathion and participates in the detoxification of malathion. These results strongly suggest that BoαE1 plays a crucial role in detoxification of malathion in B. odoriphaga .

Ovarian follicular development, follicle selection, and the process of ovulation remain poorly understood in most species. Numerous endocrine, paracrine, and autocrine factors, including the ligands represented by the transforming growth factor β (TGFβ) superfamily, TGFβ, activin, inhibin, bone morphometric protein (BMP), and growth differentiation factor (GDF) are present in the ovaries of many animals. In the present study, we investigated the immunolocalization of Smad4, a signaling molecule of the TGFβ superfamily, during folliculogenesis in the ovary of the European hedgehog (Erinaceus europaeus L., 1758). Immunolocalization studies revealed that Smad4 was widely seen in the ovary, mainly in the follicle, though its location and staining intensity varied with the different stages of the developing follicle. In the primordial follicles and early growing follicles, Smad4 protein was mainly localized in the cytoplasm of the oocyte with a half-moon staining pattern. In the pre-antral follicles, Smad4 protein was mainly located in the granulosa cells, theca cells and diffusely distributed in the interstitial cells surrounding the follicle. In the corpora lutea, the immunostaining for Smad4 was very intense. These results suggested that Smad signal transduction may play an important role in folliculogenesis and conceivably may participate in subsequent pregnancy., (Copyright © 2009 Elsevier GmbH. All rights reserved.)