Rivero-Muller, A. | Search bioRxiv

bioRxiv2020

iGIST - a kinetic bioassay for pertussis toxin based on its effect on inhibitory GPCR signaling

Detection of pertussis toxin (PTX) activity is instrumental for the development and manufacturing of pertussis vaccines. These quality and safety measures require annually thousands of mice. Here, we describe iGIST (Interference in Gi-mediated Signal Transduction) - an animal-free kinetic bioassay for detection of PTX by measuring its effect on inhibitory G protein-coupled receptor (GPCR) signaling. PTX ADP-ribosylates inhibitory -subunits of the heterotrimeric G proteins, thereby perturbing the inhibitory GPCR signaling. iGIST is based on HEK293 cells co-expressing a somatostatin receptor 2 (SSTR2), which is an inhibitory GPCR controllable by a high affinity agonist octreotide, and a luminescent 35-cyclic adenosine monophosphate (cAMP) probe. iGIST has a low sensitivity threshold in picogram/ml range of PTX, surpassing by 100-fold in a parallel analysis the currently used in vitro end-point technique to detect PTX, the cluster formation assay (CFA) in Chinese hamster ovary cells. iGIST also detects PTX in complex samples, i.e. a commercial PTX- toxoid containing pertussis vaccine that was spiked with an active PTX. iGIST has an objective digital readout and is observer-independent, offering prospects for automation. iGIST emerges as a promising animal-free alternative to detect PTX activity in the development and manufacturing of pertussis vaccines. iGIST is also expected to facilitate basic PTX research, including identification and characterization of novel compounds interfering with PTX.

microbiology↗

bioRxiv2019

Genetical engineered lung cancer cell for analyzing Epithelial-Mesenchymal transition

Cell plasticity, defined as the ability to undergo phenotypical transformation in a reversible manner, is a physiological processes that also exert important roles in disease progression Two forms of cellular plasticity are epithelial-mesenchymal transition (EMT) and its inverse process, mesenchymal-epithelial transition (MET). These processes have been correlated to the poor outcome of different types of neoplasias as well as drug resistance development. Since EMT/MET are transitional processes, we have generated and validated a reporter cell line. Specifically, a far-red fluorescent protein was knocked-in in-frame with the mesenchymal gene marker VIMENTIN (VIM) in H2170 lung cancer cells. The vimentin reporter cells (VRCs) are a reliable model for studying EMT and MET showing cellular plasticity upon a series of stimulations. These cells are a robust platform to dissect the molecular mechanisms of these processes, and for drug discovery in vitro and in the future in vivo.

bioengineering↗