A CRISPR/Cas9-Based Assay for High-Throughput Studies of Cancer-Induced Innervation.

The aggressive nature of certain cancers and their adverse effects on patient outcomes have been linked to cancer innervation, where neurons infiltrate and differentiate within the cancer stroma. Recently we demonstrated how cancer plasticity and TGFß signaling could promote breast cancer innervation that is associated with increased cancer aggressivity. Despite the promising potential of cancer innervation as a target for anti-cancer therapies, there is currently a significant lack of effective methods to study cancer-induced neuronal differentiation, hindering the development of high-throughput approaches for identifying new targets or pharmacological inhibitors against cancer innervation. To overcome this challenge, we used CRISPR-based endogenous labeling of the neuronal marker ß3-tubulin in neuronal precursors to investigate cancer-induced neuronal differentiation in nerve-cancer cocultures and provide a tool that allows for better standardization and reproducibility of studies about cancer-induced innervation. Our approach demonstrated that ß3-tubulin gene editing did not affect neuronal behavior and enabled accurate reporting of cancer-induced neuronal differentiation dynamics in high-throughput settings, which makes this approach suitable for screening large cohorts of cells or testing various biological contexts. In a more context-based approach, by combining this method with a cell model of breast cancer epithelial-mesenchymal transition, we revealed the role of cancer cell plasticity in promoting neuronal differentiation, suggesting that cancer innervation represents an underexplored path for epithelial-mesenchymal transition-mediated cancer aggressivity.

Detection of Elm Yellows Phytoplasma in Elms and Insects Using Real-Time PCR.

A rapid and accurate method to detect the common strain of elm yellows (EY) phytoplasma in elm and insect samples was developed using a real-time polymerase chain reaction (PCR) procedure based on the TaqMan minor-groove-binder probe. Primers and probe were designed based on the EY phytoplasma-specific translocation protein secY gene DNA sequence. Success of the DNA extraction procedure was evaluated by amplifying the chloroplast trnL gene of Ulmus americana. The real-time PCR assay reacted positively with EY and EY phytoplasma strain ULW DNA, an isolate which occurs in Europe. It did not cross-react with Illinois EY or aster yellows phytoplasma DNA, both of which are known to occur in elm trees in the United States, nor did it amplify several other phytoplasmas belonging to the 16SrV and other phylogenetic groups. The real-time PCR protocol was used to identify 30 EY-positive elm trees on The Pennsylvania State University, University Park campus. Threshold cycle (CT) values obtained from the EY phytoplasma-infected elm trees ranged from 15 to 37. EY phytoplasma was detected in several leafhopper taxa. This real-time PCR method can be used for the diagnostic screening of elm trees and for the identification of possible insect vectors of EY phytoplasma.