At the beginning of the project we set objectives (i) to develop a standardized workflow for using an established pull-down assay with ultra-high resolution mass spectrometry and effect-directed analysis or other bioassay for streamlined detection of compounds of effect, (ii) to use additional nuclear receptors for use on the pull down assay methodology to determine if a single workflow is applicable, and (iii) to validate the method using environmental samples of varying complexity.
The project was initiated in November 2019, and we investigated the existing equipment within the hosting department for the feasibility of protein synthesis, bulk fractionation of samples for effect-directed analysis and the method development for pull-down assays. We considered an option to use either bound protein as in a pull-down assay or potentially a dialysis option developed by a post-doctoral student at the laboratory. The project implementation was complicated by the COVIDS-19 outbreak in March 2020 causing the long-term shutdown of the Toxicological centre. During the home office period, the researcher had a chance to improve his computer programming skills in Python as a useful tool for the assessment of large data sets. He also worked on processing existing data on human exposure to synthetic compounds and published several manuscripts. Namely, he further developed a previous collaboration with the University of Cartagena co-supervising some PhD candidates and contributing to related manuscripts. The researcher also performed a literature search on compounds of emerging interest and worked on predictive software to build a spectral database for high-resolution mass spectrometry. Starting November 2020, the researcher was able to work in the laboratories under strict safety conditions. Due to circumstances, it was decided not to synthesize in-house proteins and to rely on commercial material for the project. To this end, the focus of the work returned to the initial design of trialling a PDA for the NR pregnane X receptor (PXR). After returning to Czechia, the researcher continued to pursue the project objectives.
A pull-down assay was developed using known binding compounds, and various proteins were tested. They included RAR binding retinoic acids within the cell of almost all living organisms. In mammals it is linked to cell differentiation, immune function, haematopoiesis, reproduction, vision, and embryonic patterning. In plants it is a driver of root organogenesis. Though there are concerns regarding synthetic compounds and RAR interactions there is also investigations suggesting that algal blooms may also be sources of RAR interacting compounds. TTR as the thyroxine and retinol transporter was also used. It can be secreted in the liver and enter the blood stream or in the choroid plexus and enter the cerebrospinal fluid. As TTR transports thyroid hormones any disruption in children, for instance, can impact their brain development.
The project results and outputs include (i) 2 Gold Open Access publications and 3 manuscripts, (ii) presentations at four conferences, (iii) attendance of the researcher at the RSC-IITM Desktop Seminar on Environmental Sciences series and a lecture for students at RECETOX, (iv) co-supervision of PhD students.
