Memory effects of prior subculture may impact the quality of multiomic perturbation profiles.

Mass spectrometry-based omics technologies are increasingly used in perturbation studies to map drug effects to biological pathways by identifying significant molecular events. Significance is influenced by fold change and variation of each molecular parameter, but also by multiple testing corrections. While the fold change is largely determined by the biological system, the variation is determined by experimental workflows. Here, it is shown that memory effects of prior subculture can influence the variation of perturbation profiles using the two colon carcinoma cell lines SW480 and HCT116. These memory effects are largely driven by differences in growth states that persist into the perturbation experiment. In SW480 cells, memory effects combined with moderate treatment effects amplify the variation in multiple omics levels, including eicosadomics, proteomics, and phosphoproteomics. With stronger treatment effects, the memory effect was less pronounced, as demonstrated in HCT116 cells. Subculture homogeneity was controlled by real-time monitoring of cell growth. Controlled homogeneous subculture resulted in a perturbation network of 321 causal conjectures based on combined proteomic and phosphoproteomic data, compared to only 58 causal conjectures without controlling subculture homogeneity in SW480 cells. Some cellular responses and regulatory events were identified that extend the mode of action of arsenic trioxide (ATO) only when accounting for these memory effects. Controlled prior subculture led to the finding of a synergistic combination treatment of ATO with the thioredoxin reductase 1 inhibitor auranofin, which may prove useful in the management of NRF2-mediated resistance mechanisms.

Plasma Instead of Serum Avoids Critical Confounding of Clinical Metabolomics Studies by Platelets.

Metabolomics is an emerging and powerful bioanalytical method supporting clinical investigations. Serum and plasma are commonly used without rational prioritization. Serum is collected after blood coagulation, a complex biochemical process involving active platelet metabolism. This may affect the metabolome and increase the variance, as platelet counts and function may vary substantially in individuals. A multiomics approach systematically investigating the suitability of serum and plasma for clinical studies demonstrated that metabolites correlated well (n = 461, R2 = 0.991), whereas lipid mediators (n = 83, R2 = 0.906) and proteins (n = 322, R2 = 0.860) differed substantially between specimen. Independently, analysis of platelet releasates identified most biomolecules significantly enriched in serum compared to plasma. A prospective, randomized, controlled parallel group metabolomics trial with acetylsalicylic acid administered for 7 days demonstrated that the apparent drug effects significantly differ depending on the analyzed specimen. Only serum analyses of healthy individuals suggested a significant downregulation of TXB2 and 12-HETE, which were specifically formed during coagulation in vitro. Plasma analyses reliably identified acetylsalicylic acid effects on metabolites and lipids occurring in vivo such as an increase in serotonin, 15-deoxy-PGJ2 and sphingosine-1-phosphate and a decrease in polyunsaturated fatty acids. The present data suggest that plasma should be preferred above serum for clinical metabolomics studies as the serum metabolome may be substantially confounded by platelets.

Impaired retinal oxygen metabolism and perfusion are accompanied by plasma protein and lipid alterations in recovered COVID-19 patients.

The aim of the present study was to investigate retinal microcirculatory and functional metabolic changes in patients after they had recovered from a moderate to severe acute COVID-19 infection. Retinal perfusion was quantified using laser speckle flowgraphy. Oxygen saturation and retinal calibers were assessed with a dynamic vessel analyzer. Arterio-venous ratio (AVR) was calculated based on retinal vessel diameter data. Blood plasma samples underwent mass spectrometry-based multi-omics profiling, including proteomics, metabolomics and eicosadomics. A total of 40 subjects were included in the present study, of which 29 had recovered from moderate to severe COVID-19 within 2 to 23 weeks before inclusion and 11 had never had COVID-19, as confirmed by antibody testing. Perfusion in retinal vessels was significantly lower in patients (60.6 ± 16.0 a.u.) than in control subjects (76.2 ± 12.1 a.u., p = 0.006). Arterio-venous (AV) difference in oxygen saturation and AVR was significantly lower in patients compared to healthy controls (p = 0.021 for AVR and p = 0.023 for AV difference in oxygen saturation). Molecular profiles demonstrated down-regulation of cell adhesion molecules, NOTCH3 and fatty acids, and suggested a bisphasic dysregulation of nitric oxide synthesis after COVID-19 infection. The results of this study imply that retinal perfusion and oxygen metabolism is still significantly altered in patients well beyond the acute phase of COVID-19. This is also reflected in the molecular profiling analysis of blood plasma, indicating a down-regulation of nitric oxide-related endothelial and immunological cell functions.Trial Registration: ClinicalTrials.gov ( https://clinicaltrials.gov ) NCT05650905.