Matrix-Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging of Metabolites during Sorghum Germination.

Dhurrin is the most abundant cyanogenic glucoside found in sorghum (Sorghum bicolor) where it plays a key role in chemical defense by releasing toxic hydrogen cyanide upon tissue disruption. Besides this well-established function, there is strong evidence that dhurrin plays additional roles, e.g. as a transport and storage form of nitrogen, released via endogenous recycling pathways. However, knowledge about how, when and why dhurrin is endogenously metabolized is limited. We combined targeted metabolite profiling with matrix-assisted laser desorption/ionization-mass spectrometry imaging to investigate accumulation of dhurrin, its recycling products and key general metabolites in four different sorghum lines during 72 h of grain imbibition, germination and early seedling development, as well as the spatial distribution of these metabolites in two of the lines. Little or no dhurrin or recycling products were present in the dry grain, but their de novo biosynthesis started immediately after water uptake. Dhurrin accumulation increased rapidly within the first 24 h in parallel with an increase in free amino acids, a key event in seed germination. The trajectories and final concentrations of dhurrin, the recycling products and free amino acids reached within the experimental period were dependent on genotype. Matrix-assisted laser desorption/ionization-mass spectrometry imaging demonstrated that dhurrin primarily accumulated in the germinating embryo, confirming its function in protecting the emerging tissue against herbivory. The dhurrin recycling products, however, were mainly located in the scutellum and/or pericarp/seed coat region, suggesting unknown key functions in germination.

Impedimetric toxicity assay in microfluidics using free and liposome-encapsulated anticancer drugs.

In this work, we have developed a microfluidic cytotoxicity assay for a cell culture and detection platform, which enables both fluid handling and electrochemical/optical detection. The cytotoxic effect of anticancer drugs doxorubicin (DOX), oxaliplatin (OX) as well as OX-loaded liposomes, developed for targeted drug delivery, was evaluated using real-time impedance monitoring. The time-dependent effect of DOX on HeLa cells was monitored and found to have a delayed onset of cytotoxicity in microfluidics compared with static culture conditions based on data obtained in our previous study. The result of a fluorescent microscopic annexin V/propidium iodide assay, performed in microfluidics, confirmed the outcome of the real-time impedance assay. In addition, the response of HeLa cells to OX-induced cytotoxicity proved to be slower than toxicity induced by DOX. A difference in the time-dependent cytotoxic response of fibrosarcoma cells (HT1080) to free OX and OX-loaded liposomes was observed and attributed to incomplete degradation of the liposomes, which results in lower drug availability. The matrix metalloproteinase (MMP)-dependent release of OX from OX-loaded liposomes was also confirmed using laryngopharynx carcinoma cells (FaDu). The comparison and the observed differences between the cytotoxic effects under microfluidic and static conditions highlight the importance of comparative studies as basis for implementation of microfluidic cytotoxic assays.