Chemical Composition of Essential Oil of Ageratum conyzoides with Antifungal Activity on the Lasiodiplodia theobromae Strain in the Region of Kisangani and DR Congo

Aims: To determine the chemical composition of essential oil of Ageratum conyzoides with antifungal activity on the strain of Lasiodiplodia theobromae in the Kisangani region. Location and Duration of Studies: Faculty of Sciences of University of Kisangani (Democratic Republic of Congo) and Faculty of Biosciences Engineering of University of Ghent (Belgium), between May to November 2019. Methodology: The essential oil from the leaves of A. conyzoides was extracted by hydrodistillation. Potato dextrose agar (PDA) was used as a culture medium. In vitro evaluation of its antifungal activity was performed on PDA medium and expressed as percentage of inhibition (PI). Results: The extracted essential oil (with a yield of 0.63%) showed a PI of 91.63% on the strain of L. theobromae after two days of incubation. It consists of at least 23 compounds, of which seven are in the majority (abundances greater than 1.5% and represent 92.05%), namely Precocene I (38.33%), Beta-caryophyllene (26.51%), Beta-sesquiphellandrene (8.63%), Beta-cubebene (7.91%), Alpha-muurolene (4.95%), 1.5.9.9-Tetramethyl-1.4.7-cycloundecatriene (3.04%), Cis-beta-farnesene (2.99%). The hydrocarbon sesquiterpenes are the most frequent compounds (58.95%). Discussion: Compared to the A. conyzoides saponins the PI obtained from the essential oil is higher. The essential oil chemotype obtained from the leaves of A. conyzoides is of the Precocene I type, similar to the oils from the stems and flowers of the same plant in West Africa. Conclusion: The essential oil of A. conyzoides has a very high inhibitory power on L. theobromae. It mainly contains the Precocene and the hydrocarbon sesquiterpenes. Assessment of the antifungal activity of each separate molecule should be considered.

Optimisation of Widefield Fluorescence Fret System for Studying Separate Molecule Interactions

The Förster Resonance Energy Transfer (FRET) method has wide application in modern science for studying protein–protein interactions and conformational changes. FRET allows to assess molecular interactions by measuring energy transfer between acceptor and donor fluorophores coupled to the molecule(s) of interest. The method demands high precision in experimental design, experimental settings and correct data interpretation. Therefore, we tested several parameters to estimate FRET measurement accuracy in our Nikon wide-field fluorescence FRET system. The experiments were performed in a HEK-293 cell line transfected with DNA constructs expressing Calcium Release-Activated Channel (CRAC) subunits STIM1 and ORAI1 coupled to donor fluorophore Cyan Fluorescent Protein (CFP) and acceptor fluorophore Yellow Fluorescent Protein (YFP), respectively. Exposure time and approach of data analysis varied throughout experiments in order to optimise FRET data quality. Dependence of FRETeff values on measurement quality and donor/acceptor fluorophore ratio in the cells was estimated. We demonstrated that, using the wide-field fluorescence FRET system, minimising the exposure of fluorophores before measurement using neutral density (ND) filters considerably minimises undesirable photo-bleaching of the fluorophores. There was a strong correlation between the CFP/YFP ratio in the cells and the observed FRET level, suggesting that only cells with certain donor/acceptor ratio might be comparable. We also showed impact of FRET measurement quality, defined as accordance of FRET pixels to Gaussian distribution, on FRET artefacts. Knowledge obtained during our experiments may be important for approbating similar wide-field fluorescence FRET systems to study two separate molecule interactions and for understanding the correct setup of the experiments and data interpretation.