The early metabolic signatures associated with the progression of septic shock and with responsiveness to therapy can be useful for developing target therapy. The Sequential Organ Failure Assessment (SOFA) score is used for stratifying risk and predicting mortality. This study aimed to verify whether different responses to therapy, assessed as changes in SOFA score at admission (T1, acute phase) and 48 h later (T2, post-resuscitation), are associated with different metabolite patterns. We examined the plasma metabolome of 21 septic shock patients (pts) enrolled in the Shockomics clinical trial (NCT02141607). Patients for which SOFAT2 was >8 and Δ = SOFAT1 − SOFAT2 < 5, were classified as not responsive to therapy (NR, 7 pts), the remaining 14 as responsive (R). We combined untargeted and targeted mass spectrometry-based metabolomics strategies to cover the plasma metabolites repertoire as far as possible. Metabolite concentration changes from T1 to T2 (Δ = T2 − T1) were used to build classification models. Our results support the emerging evidence that lipidome alterations play an important role in individual patients’ responses to infection. Furthermore, alanine indicates a possible alteration in the glucose-alanine cycle in the liver, providing a different picture of liver functionality from bilirubin. Understanding these metabolic disturbances is important for developing any effective tailored therapy for these patients.
Studies of cellular internalization of nanoparticles (NPs) play a paramount role for the design of efficient drug delivery systems, but so far they lack a robust experimental technique able to quantify the NP uptake in terms of number of NPs internalized in each cell. In this work we propose a novel method which provides a quantitative evaluation of fluorescent NP uptake by combining flow cytometry and plate fluorimetry with measurements of number of cells. Single cell fluorescence signals measured by flow cytometry were associated with the number of internalized NPs, exploiting the observed linearity between average flow cytometric fluorescence and overall plate fluorimeter measures, and previous calibration of the microplate reader with serial dilutions of NPs. This precise calibration has been made possible by using biocompatible fluorescent NPs in the range of 20-300 nm with a narrow particle size distribution, functionalized with a covalently bonded dye, Rhodamine B, and synthesized via emulsion free-radical polymerization. We report the absolute number of NPs internalized in mouse mammary tumor cells (4T1) as a function of time for different NP dimensions and surface charges and at several exposure concentrations. The obtained results indicate that 4T1 cells incorporated 10(3)-10(4) polymer NPs in a short time, reaching an intracellular concentration 15 times higher than the external one.
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