Browsing by Issue Date, starting with "2020-04-09"
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- A Simple, label-free, and high-throughput method to evaluate the epigallocatechin-3-gallate impact in plasma molecular profilePublication . Araújo, Rúben; Ramalhete, Luís; Da Paz, Helder; Ribeiro, Edna; Calado, CecíliaEpigallocatechin-3-gallate (EGCG), the major catechin presente in green tea, presents diverse appealing biological activities, such as antioxidative, anti-inflammatory, antimicrobial, and antiviral activities, among others. The present work evaluated the impact in the molecular profile of human plasma from daily consumption of 225 mg of EGCG for 90 days. Plasma from peripheral blood was collected from 30 healthy human volunteers and analyzed by high-throughput Fourier transform infrared spectroscopy. To capture the biochemical information while minimizing the interference of physical phenomena, several combinations of spectra pre-processing methods were evaluated by principal component analysis. The pre-processing method that led to the best class separation, that is, between the plasma spectral data collected at the beginning and after the 90 days, was a combination of atmospheric correction with a second derivative spectra. A hierarchical cluster analysis of second derivativespectraalsohighlightedthefactthatplasmaacquiredbeforeEGCGconsumptionpresented a distinct molecular profile after the 90 days of EGCG consumption. It was also possible by partial least squares regression discriminant analysis to correctly predict all unlabeled plasma samples (not used for model construction) at both timeframes. We observed that the similarity in composition among the plasma samples was higher in samples collected after EGCG consumption when compared with the samples taken prior to EGCG consumption. Diverse negative peaks of the normalized second derivative spectra, associated with lipid and protein regions, were significantly affected (p < 0.001) by EGCG consumption, according to the impact of EGCG consumption on the patients’ blood, low density and high density lipoproteins ratio. In conclusion, a single bolus dose of 225 mg of EGCG, ingested throughout a period of 90 days, drastically affected plasma molecular composition in all participants, which raises awareness regarding prolonged human exposure to EGCG. Because the analysis was conducted in a high-throughput, label-free, and economic analysis, it could be applied to high-dimension molecular epidemiological studies to further promote the understanding of the effect of bio-compound consumption mode and frequency.
- Metabolic fingerprinting with Fourier-Transform Infrared (FTIR) spectroscopy: towards a high-throughput screening assay for antibiotic discovery and mechanism-of-action elucidationPublication . Ribeiro Da Cunha, Bernardo; Fonseca, Luís P. P.; Calado, CecíliaThe discovery of antibiotics has been slowing to a halt. Phenotypic screening is once again at the forefront of antibiotic discovery, yet Mechanism-Of-Action (MOA) identification is still a major bottleneck. As such, methods capable of MOA elucidation coupled with the high-throughput screening of whole cells are required now more than ever, for which Fourier-Transform Infrared (FTIR) spectroscopy is a promising metabolic fingerprinting technique. A high-throughput whole-cell FTIR spectroscopy-based bioassay was developed to reveal the metabolic fingerprint induced by 15 antibiotics on the Escherichia coli metabolism. Cells were briefly exposed to four times the minimum inhibitory concentration and spectra were quickly acquired in the high-throughput mode. After preprocessing optimization, a partial least squares discriminant analysis and principal component analysis were conducted. The metabolic fingerprints obtained with FTIR spectroscopy were sufficiently specific to allow a clear distinction between different antibiotics, across three independent cultures, with either analysis algorithm. These fingerprints were coherent with the known MOA of all the antibiotics tested, which include examples that target the protein, DNA, RNA, and cell wall biosynthesis. Because FTIR spectroscopy acquires a holistic fingerprint of the effect of antibiotics on the cellular metabolism, it holds great potential to be used for high-throughput screening in antibiotic discovery and possibly towards a better understanding of the MOA of current antibiotics.