an-integrated-raman-spectroscopy-mass-spectrometry-platform-to-study

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Biodynamics Research Center (BDR), RIKEN

This study aimed to analyse the prevalence and genetic diversity of the tet(M) resistance gene as well as the species composition of oxytetracycline-resistant bacteria in coastal areas of South Korea. Both culturable and non-culturable bacterial communities were sampled in 2010 and 2011 within two coastal areas (Wando and Geoje). tet(M) and 16S rRNA gene sequences were obtained by PCR and sequencing and were used for phylogenetic analyses. Quantitative PCR (qPCR) was performed to determine the prevalence of tet(M) in sampled areas. Phylogenetic analyses revealed high heterogeneity of tet(M) sequences between Wando and Geoje areas. Sequences found in Wando were highly similar to each other and were similar to sequences found in Japanese aquaculture sites. A larger diversity of tet(M) sequences was obtained from natural assemblage in Geoje. qPCR showed a high prevalence of tet(M) in Wando's aquaculture sites (up to 10gene copies per 16S rRNA copy) and for industrial sites in Geoje (up to 10gene copies per 16S rRNA copy). 16 rRNA-based identification showed that Vibrio sp. and Photobacterium sp. were the most representative species in Wando, whereas in Geoje a broader range of species was found, with several isolates identified as pathogenic Acinetobacter, Aeromonas and Klebsiella. Altogether, these results showed a high prevalence of tet(M) in both sites, and different origins of contamination were identified. Because several pathogenic strains of oxytetracycline-resistant bacteria were reported for the first time, these results strongly warrant further analyses and actions to prevent contamination events by antibiotics in South Korea.

Genetic diversity of oxytetracycline-resistant bacteria and tet(M) genes in two major coastal areas of South Korea.

Monitoring drug uptake, its metabolism, and response on the single-cell level is invaluable for sustaining drug discovery efforts. In this study, we show the possibility of accessing the information about the aforementioned processes at the single-cell level by monitoring the anticancer drug tamoxifen using live single-cell mass spectrometry (LSC-MS) and Raman spectroscopy. First, we explored whether Raman spectroscopy could be used as a label-free and nondestructive screening technique to identify and predict the drug response at the single-cell level. Then, a subset of the screened cells was isolated and analyzed by LSC-MS to measure tamoxifen and its metabolite, 4-Hydroxytamoxifen (4-OHT) in a highly selective, sensitive, and semiquantitative manner. Our results show the Raman spectral signature changed in response to tamoxifen treatment which allowed us to identify and predict the drug response. Tamoxifen and 4-OHT abundances quantified by LSC-MS suggested some heterogeneity among single-cells. A similar phenomenon was observed in the ratio of metabolized to unmetabolized tamoxifen across single-cells. Moreover, a correlation was found between tamoxifen and its metabolite, suggesting that the drug was up taken and metabolized by the cell. Finally, we found some potential correlations between Raman spectral intensities and tamoxifen abundance, or its metabolism, suggesting a possible relationship between the two signals. This study demonstrates for the first time the potential of using Raman spectroscopy and LSC-MS to investigate pharmacokinetics at the single-cell level.

Arno Germond

Biodynamics Research Center (BDR), RIKEN

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects