Identification of compounds inhibiting prion replication and toxicity by removing PrPC from the cell surface.

The vast majority of therapeutic approaches tested so far for prion diseases, transmissible neurodegenerative disorders of human and animals, tackled PrPSc , the aggregated and infectious isoform of the cellular prion protein (PrPC ), with largely unsuccessful results. Conversely, targeting PrPC expression, stability or cell surface localization are poorly explored strategies. We recently characterized the mode of action of chlorpromazine, an anti-psychotic drug known to inhibit prion replication and toxicity by inducing the re-localization of PrPC from the plasma membrane. Unfortunately, chlorpromazine possesses pharmacokinetic properties unsuitable for chronic use in vivo, namely low specificity and high toxicity. Here, we employed HEK293 cells stably expressing EGFP-PrP to carry out a semi-automated high content screening (HCS) of a chemical library directed at identifying non-cytotoxic molecules capable of specifically relocalizing PrPC from the plasma membrane as well as inhibiting prion replication in N2a cell cultures. We identified four candidate hits inducing a significant reduction in cell surface PrPC , one of which also inhibited prion propagation and toxicity in cell cultures in a strain-independent fashion. This study defines a new screening method and novel anti-prion compounds supporting the notion that removing PrPC from the cell surface could represent a viable therapeutic strategy for prion diseases.

A High-Content Screening of Anticancer Compounds Suggests the Multiple Tyrosine Kinase Inhibitor Ponatinib for Repurposing in Neuroblastoma Therapy.

Novel druggable targets have been discovered in neuroblastoma (NB), paving the way for more effective treatments. However, children with high-risk NB still show high mortality rates prompting for a search of novel therapeutic options. Here, we aimed at repurposing FDA-approved drugs for NB treatment by performing a high-content screening of a 349 anticancer compounds library. In the primary screening, we employed three NB cell lines, grown as three-dimensional (3D) multicellular spheroids, which were treated with 10 µmol/L of the library compounds for 72 hours. The viability of 3D spheroids was evaluated using a high-content imaging approach, resulting in a primary hit list of 193 compounds. We selected 60 FDA-approved molecules and prioritized drugs with multi-target activity, discarding those already in use for NB treatment or enrolled in NB clinical trials. Hence, 20 drugs were further tested for their efficacy in inhibiting NB cell viability, both in two-dimensional and 3D models. Dose-response curves were then supplemented with the data on side effects, therapeutic index, and molecular targets, suggesting two multiple tyrosine kinase inhibitors, ponatinib and axitinib, as promising candidates for repositioning in NB. Indeed, both drugs showed induction of cell-cycle block and apoptosis, as well as inhibition of colony formation. However, only ponatinib consistently affected migration and inhibited invasion of NB cells. Finally, ponatinib also proved effective inhibition of tumor growth in orthotopic NB mice, providing the rationale for its repurposing in NB therapy. Mol Cancer Ther; 17(7); 1405-15. ©2018 AACR.

Biomolecular identification of allergenic pollen: a new perspective for aerobiological monitoring?

BACKGROUND: Accurate and updated information on airborne pollen in specific areas can help allergic patients. Current monitoring systems are based on a morphologic identification approach, a time-consuming method that may represent a limiting factor for sampling network enhancement. OBJECTIVE: To verify the feasibility of developing a real-time polymerase chain reaction (PCR) approach, an alternative to optical analysis, as a rapid, accurate, and automated tool for the detection and quantification of airborne allergenic pollen taxa. METHODS: The traditional cetyl trimethyl ammonium bromide-based method was modified for DNA isolation from pollen. Taxon-specific DNA sequences were identified via bioinformatics or literature searches and were PCR amplified from the matching allergenic taxa; based on the sequences of PCR products, complementary or degenerate TaqMan probes were developed. The accuracy of the quantitative real-time PCR assay was tested on 3 plant species. RESULTS: The setup of a modified DNA extraction protocol allowed us to achieve good-quality pollen DNA. Taxon-specific nuclear gene fragments were identified and sequenced. Designed primer pairs and probes identified selected pollen taxa, mostly at the required classification level. Pollen was properly identified even when collected on routine aerobiological tape. Preliminary quantification assays on pollen grains were successfully performed on test species and in mixes. CONCLUSIONS: The real-time PCR approach revealed promising results in pollen identification and quantification, even when analyzing pollen mixes. Future perspectives could concern the development of multiplex real-time PCR for the simultaneous detection of different taxa in the same reaction tube and the application of high-throughput molecular methods.

Isolation of functional RNA from small amounts of different grape and apple tissues.

An efficient, simple, and small-scale procedure for isolating functional ribonucleic acid (RNA) was successfully applied to many different tissues of grape and apple. These woody plants are rich in polyphenolic compounds and polysaccharides that could impair the RNA extraction. The method chosen is based on the use of hot borate buffer at alkaline pH supplemented with several adjuvants and followed by selective precipitations. Starting with only 0.4 g of fresh tissue and working with small tubes (2 mL), we were able to obtain good yields of high-quality RNA suitable for further applications. The procedure can be proposed for many applications, and it is particularly highly recommended when isolating RNA from a large number of samples.