Additional file 3: Figure S2. of Retinoid and carotenoid status in serum and liver among patients at high-risk for liver cancer

Boxplots (10th and 90th Percentile) of urinary isoprostanes by fibrosis stage. (EPS 102 kb

Additional file 1: Table S1. of Retinoid and carotenoid status in serum and liver among patients at high-risk for liver cancer

Clinical assay results for the study population by fibrosis stage - median (interquartile range). (DOCX 38 kb

Psychosine increases rigidity in focalized areas of the RBC plasma membrane.

<p><b>A-C)</b> Overall fluidity in RBCs (<b>A,B</b>) and purified P40 brain myelin (<b>C</b>) measured by TMA-DPH is not affected by psychosine or in Twitcher mice. Wild-type, Twitcher, and wild-type RBCs preincubated with increasing concentrations of psychosine were analyzed by TMA-DPH anisotropy. <b>D-H)</b> Multiphoton excitation microscopy imaging of Laurdan in P40 wild-type (<b>D</b>), Twitcher (<b>E</b>), and wild-type RBCs treated with 2 (<b>F</b>), 5 (<b>G</b>) or 10 μM of psychosine for 30 minutes at 37^°C. (<b>H</b>). GP images are in pseudo-colors with the range indicated by the color bar going from fluid (blue/green) to rigid (yellow/red). Scale bar, 10 μm. <b>I)</b> GP value from the center of RBCs were analyzed as represented inside the white dashed circles in (<b>D</b>) and plotted as % of change from wild-type values. <b>J</b>) Distribution of domains of high GP and high-rigidity. Results are mean ± SEM of samples N = 68–257 in 3–4 independent experiments. NS, not significant (>0.05); *, <i>p</i> <0.05; **, <i>p</i> <0.01; ***, <i>p</i> <0.001.</p

Psychosine enhances the shedding of membrane microvesicles: Implications in demyelination in Krabbe’s disease

<div><p>In prior studies, our laboratory showed that psychosine accumulates and disrupts lipid rafts in brain membranes of Krabbe’s disease. A model of lipid raft disruption helped explaining psychosine’s effects on several signaling pathways important for oligodendrocyte survival and differentiation but provided more limited insight in how this sphingolipid caused demyelination. Here, we have studied how this cationic inverted coned lipid affects the fluidity, stability and structure of myelin and plasma membranes. Using a combination of cutting-edge imaging techniques in non-myelinating (red blood cell), and myelinating (oligodendrocyte) cell models, we show that psychosine is sufficient to disrupt sphingomyelin-enriched domains, increases the rigidity of localized areas in the plasma membrane, and promotes the shedding of membranous microvesicles. The same physicochemical and structural changes were measured in myelin membranes purified from the mutant mouse Twitcher, a model for Krabbe’s disease. Areas of higher rigidity were measured in Twitcher myelin and correlated with higher levels of psychosine and of myelin microvesiculation. These results expand our previous analyses and support, for the first time a pathogenic mechanism where psychosine’s toxicity in Krabbe disease involves deregulation of cell signaling not only by disruption of membrane rafts, but also by direct local destabilization and fragmentation of the membrane through microvesiculation. This model of membrane disruption may be fundamental to introduce focal weak points in the myelin sheath, and consequent diffuse demyelination in this leukodystrophy, with possible commonality to other demyelinating disorders.</p></div

Psychosine reduces sphingomyelin and cholesterol lateral mobility.

<p>Psychosine reduces sphingomyelin and cholesterol lateral mobility.</p

Psychosine reduces sphingomyelin lateral mobility.

<p><b>A-C)</b> Delayed time to recover from photobleaching by BODIPY-SM (<b>A</b>), BODIPY-PC (<b>B</b>) and BODIPY-GM1 (<b>C</b>) (N = 61–648 in 1–4 independent experiments). <b>D-F)</b> The mobile fraction of BODIPY-SM (<b>D</b>) but not that of BODIPY-PC (<b>E</b>) and BODIPY-GM1 (<b>F</b>) in Twitcher and wild-type RBCs treated with psychosine were significantly lower than wild-type RBCs. <b>G-I)</b> Similarly, the half-life of FRAP signal was increased for BODIPY-SM (<b>G</b>) but not for BODIPY-PC (<b>H</b>) and BODIPY-GM1 (<b>I</b>) in Twitcher and wild-type RBCs treated with psychosine, evidencing an effect of psychosine in the plasma membrane and an interaction with sphingomyelin. All aforementioned psychosine incubations were 30 minutes at 37^°C Results are mean ± SEM. NS, not significant (>0.05); *, <i>p</i> <0.05; **, <i>p</i> <0.01; ***, <i>p</i> <0.001.</p

<i>K</i>‑Targeted Metabolomic Analysis Extends Chemical Subtraction to DESIGNER Extracts: Selective Depletion of Extracts of Hops (Humulus lupulus)

This study introduces a flexible and compound targeted approach to <u>D</u>eplete and <u>E</u>nrich <u>S</u>elect <u>I</u>ngredients to <u>G</u>enerate <u>N</u>ormalized <u>E</u>xtract <u>R</u>esources, generating DESIGNER extracts, by means of chemical subtraction or augmentation of metabolites. Targeting metabolites based on their liquid–liquid partition coefficients (<i>K</i> values), <i>K</i> targeting uses countercurrent separation methodology to remove single or multiple compounds from a chemically complex mixture, according to the following equation: DESIGNER extract = total extract ± target compound(s). Expanding the scope of the recently reported depletion of extracts by immunoaffinity or solid phase liquid chromatography, the present approach allows a more flexible, single- or multi-targeted removal of constituents from complex extracts such as botanicals. Chemical subtraction enables both chemical and biological characterization, including detection of synergism/antagonism by both the subtracted targets and the remaining metabolite mixture, as well as definition of the residual complexity of all fractions. The feasibility of the DESIGNER concept is shown by <i>K</i>-targeted subtraction of four bioactive prenylated phenols, isoxanthohumol (<b>1</b>), 8-prenylnaringenin (<b>2</b>), 6-prenylnaringenin (<b>3</b>), and xanthohumol (<b>4</b>), from a standardized hops (Humulus lupulus L.) extract using specific solvent systems. Conversely, adding <i>K</i>-targeted isolates allows enrichment of the original extract and hence provides an augmented DESIGNER material. Multiple countercurrent separation steps were used to purify each of the four compounds, and four DESIGNER extracts with varying depletions were prepared. The DESIGNER approach innovates the characterization of chemically complex extracts through integration of enabling technologies such as countercurrent separation, <i>K</i>-by-bioactivity, the residual complexity concepts, as well as quantitative analysis by ¹H NMR, LC-MS, and HiFSA-based NMR fingerprinting

Psychosine affects cell surface area by vesiculation.

<p><b>A)</b> RBCs from P40 TWI or WT pre-incubated with psychosine for 30 minutes at 37^°C were labeled with BODIPY-SM, plated on poly-lysine coated slides and their surface area was measured by confocal microscopy. Twitcher cell surface area was significantly decreased, an effect also elicited in wild-type cells after exposure to 2–10 μM of psychosine (N = 28–44 in 3 independent experiments). <b>B)</b> RBCs labeled with BODIPY-SM were exposed to psychosine during a 60 min on stage incubation. Vesicles appeared at the surface following different stages of budding, from membrane outward bending (#1, #2), to small (1–2 μm, #3, #4) and large (2–4 μm #5 to #7) vesicle budding. <b>C-E)</b> Flow cytometry graphs plotting side (SSC) and forward scatter (FSC) of events with a gate selected for size 1–4 μm (microvesicles, mv) and 6–7 μm (RBCs) (C) showed a significant increase in Twitcher with respect to wild-type cells (D). Analysis of RBC CD235a confirmed the erythrocyte origin of the vesicles (expressed as erythrocyte-derived particles, EdP in E). Results in D and E were normalized by total RBCs counted in the 6–7 μm region from 3 independent experiments. Results are mean ± SEM. NS, not significant (>0.05); *, <i>p</i> <0.05; **, <i>p</i> <0.01; ***, <i>p</i> <0.001.</p

Psychosine destabilizes submicrometric lipid domains in red blood cells.

<p><b>A)</b> Red blood cells (RBCs) from Twitcher mice and healthy littermates collected at different days of age (P14-P40) were analyzed for psychosine content. Psychosine in Twitcher RBCs was significantly higher at all ages as compared to healthy control, with psychosine levels increasing with age (n = 3 in 4 independent experiments). <b>B)</b> Wild-type RBCs incubated with 0–10 μM of psychosine, washed and processed for psychosine extraction and quantification. Data from 6 independent experiments. <b>C)</b> Hemolysis was measured after incubation with 0–20 μM psychosine. <b>D, E)</b> Confocal images of P40 RBCs labeled with BODIPY-SM showed a decrease of BODIPY-SM domains in Twitcher (<b>D</b>) as compared to wild-type (<b>E</b>). Scale bars, 2 μm. <b>F)</b> Isolated-RBCs collected from P14-P40 were dyed with BODIPY-SM, -PC and -GM1 and labeled sub-micrometric domains were counted by confocal microscopy. There was no difference in domains of Twitcher <i>vs</i> wild-type RBCs from mice below 30 days of age, while domains were significantly decreased in Twitcher RBCs at P30 and P40 (N = 109–924 in 3–6 independent experiments). <b>G)</b> Domains were counted in P40 wild-type hemi-RBCs after exposure to 0–5 μM of psychosine and labeled with BODIPY-SM, -PC or -GM1. <b>H)</b> RBCs were incubated with various concentrations of psychosine before labeling with BODIPY-SM and membrane fluorescence determined. <b>I)</b> RBCs were incubated with various concentration of psychosine. Sphingomyelin (SM) and phosphatidylcholine (PC) were extracted and separated on thin layer chromatography and quantified by densitometry. All aforementioned psychosine incubations were 30 minutes at 37^°C. The results are mean ± SEM. NS, not significant (>0.05); *, <i>p</i> <0.05; **, <i>p</i> <0.01; ***, <i>p</i> <0.001.</p