Lung tumor discrimination by deep neural network model CanDo via DNA methylation in bronchial lavage.

Bronchoscopic-assisted discrimination of lung tumors presents challenges, especially in cases with contraindications or inaccessible lesions. Through meta-analysis and validation using the HumanMethylation450 database, this study identified methylation markers for molecular discrimination in lung tumors and designed a sequencing panel. DNA samples from 118 bronchial washing fluid (BWF) specimens underwent enrichment via multiplex PCR before targeted methylation sequencing. The Recursive Feature Elimination Cross-Validation and deep neural network algorithm established the CanDo classification model, which incorporated 11 methylation features (including 8 specific to the TBR1 gene), demonstrating a sensitivity of 98.6% and specificity of 97.8%. In contrast, bronchoscopic rapid on-site evaluation (bronchoscopic-ROSE) had lower sensitivity (87.7%) and specificity (80%). Further validation in 33 individuals confirmed CanDo's discriminatory potential, particularly in challenging cases for bronchoscopic-ROSE due to pathological complexity. CanDo serves as a valuable complement to bronchoscopy for the discriminatory diagnosis and stratified management of lung tumors utilizing BWF specimens.

Enhancing alfalfa resistance to Spodoptera herbivory by sequestering microRNA396 expression.

KEY MESSAGE: Sequestering microRNA396 by overexpression of MIM396 enhanced alfalfa resistance to Spodoptera litura larvae, which may be due to increased lignin content and enhanced low-molecular weight flavonoids and glucosinolates biosynthesis. Alfalfa (Medicago sativa), the most important leguminous forage crop, suffers from the outbreak of defoliator insects, especially Spodoptera litura, resulting in heavy losses in yield and forage quality. Here, we found that the expression of alfalfa microRNA396 (miR396) precursor genes and mature miR396 was significantly up-regulated in wounding treatment that simulates feeding injury by defoliator insects. To verify the function of miR396 in alfalfa resistance to insect, we generated MIM396 transgenic alfalfa plants with significantly down-regulated miR396 expression by Agrobacterium-mediated genetic transformation. The MIM396 transgenic alfalfa plants exhibited improved resistance to Spodoptera litura larvae with increased lignin content but decreased JA accumulation. Most of the miR396 putative target GRF genes were up-regulated in MIM396 transgenic lines, and responded to the wounding treatment. By RNA sequencing analysis, we found that the differentially expressed genes related to insect resistance between WT and MIM396 transgenic plants mainly clustered in biosynthesis pathways in lignin, flavonoids and glucosinolates. In addition to the phenotype of enhanced insect resistance, MIM396 transgenic plants also displayed reduced biomass yield and forage quality. Our results broaden the function of miR396 in alfalfa and provide genetic resources for studying alfalfa insect resistance.