Characterization and isolation of a large population of cells are indispensable procedures in biological sciences. Flow cytometry is one of the standards that offers a method to characterize and isolate cells at high throughput. When performing flow cytometry, cells are molecularly stained with fluorescent labels to adopt biomolecular specificity which is essential for characterizing cells. However, molecular staining is costly and its chemical toxicity can cause side effects to the cells which becomes a critical issue when the cells are used downstream as medical products or for further analysis. Here, we introduce a high-throughput stain-free flow cytometry called in silico-labeled ghost cytometry which characterizes and sorts cells using machine-predicted labels. Instead of detecting molecular stains, we use machine learning to derive the molecular labels from compressive data obtained with diffractive and scattering imaging methods. By directly using the compressive ‘imaging’ data, our system can accurately assign the designated label to each cell in real time and perform sorting based on this judgment. With this method, we were able to distinguish different cell states, cell types derived from human induced pluripotent stem (iPS) cells, and subtypes of peripheral white blood cells using only stain-free modalities. Our method will find applications in cell manufacturing for regenerative medicine as well as in cell-based medical diagnostic assays in which fluorescence labeling of the cells is undesirable.
The permeation of ebiratide (H-Met(O2)-Glu-His-Phe-D-Lys-Phe-NH(CH2)8NH2), a novel ACTH analogue, across the intestinal mucosae has been examined by use of isolated intestinal membranes from rats in a modified Ussing chamber. Regional differences were observed in the permeation of ebiratide across intestinal membranes; the order of membrane permeability was jejunum > ileum > duodenum > colon. Overall, the permeation of ebiratide was relatively poor. The effects of various absorption enhancers were examined to increase the intestinal permeability to ebiratide. Sodium glycocholate and sodium caprate had no significant enhancing effect on the permeability of the jejunal membrane, but significantly enhanced the permeation of ebiratide through the colonic membrane. On the other hand, N-dodecyl-beta-D-maltopyramoside (LM) significantly enhanced the permeation of ebiratide through both jejunal and colonic membranes. In general, the absorption-enhancing effects of these agents were more predominant in the colon than in the jejunum. Membrane damage by the absorption enhancers was evaluated by measuring the amount of protein released from the intestinal membrane. It was found that all the absorption enhancers slightly increased the amount of protein released, but that the amounts of protein released in the presence of these enhancers were much less than in the presence of ethylenediaminetetraacetic acid (EDTA), used as a positive control. These findings suggest that the absorption enhancers, especially LM might be useful adjuvants for improving the intestinal absorption of peptide and protein drugs, including ebiratide.
A stereoselective and sensitive method for the determination of the enantiomers of felodipine, a dihydropyridine calcium antagonist, has been developed and the pharmacokinetic profiles of the enantiomers comparatively studied after oral administration to dogs and humans. D6-Felodipine, the internal standard, was added to the plasma, extracted with a solvent and then optically resolved into S(-) and R(+) enantiomers on a high performance liquid chromatographic Chiralcel OJ column. Each enantiomer in the effluent was analysed by capillary column gas chromatography/positive ion electron impact mass spectrometry. After oral administration of the felodipine racemate, the Tmax and t1/2 values hardly differed between the two enantiomers in dogs and humans. The Cmax and AUC0-24 h values of the S(-) enantiomer were slightly higher than those of the R(+) enantiomer in humans but the difference between the enantiomers was not significant. These results suggested that there is no large difference in the absorption, distribution and elimination of felodipine enantiomers after oral administration of the racemate in either dog or human.
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