Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Characterization of EVs Isolated from Fetal Mouse Neurosphere Cultures 2A) Transmission electron micrograph images of CD63-immunogold-labelled EVs. Red circles indicate immunogold puncta, indicating CD63-like immunoreactivity. 2B) Frequency Distribution of EV diameter. Inset depicts Nanosight image of EVs derived from NSCs. 2C) Quantitative immunoblot analysis of relative protein levels of positive EV markers Tsg101, AnnexinVI, Rab5, CD63, CD9. 2D) negative EV markers Calnexin, Cytochrome C, Ago2 from NSCs and isolated EVs from the culture media of these NSCs. n = 3 to 7 samples per group. Significance was determined using unpaired t-test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, # p < 0.08. 2E) Representative immunoblots of positive EV markers, Tsg101 and annexin VI (green fluorescence). 2F) Representative immunoblots of negative EV markers, Ago2 and Calnexin (green fluorescence). Red bars at the base of each immunoblot denote cell lysates (CL) and green bars denote EV-derived protein lysates (EV).

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Isolation, Transmission Assay, Derivative Assay, Western Blot, Fluorescence

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: WGCNA-based Comparisons of the Protein Content of EVs Relative to Parental NSCs. WGCNA of proteins expressed in all 36 samples, shows that EVs and their parent cells have distinct protein networks and correlations with the trait of “location” (Cell vs. EV) contributing to the majority of the difference between clusters. Other traits including “pregnancy” (cells derived from three separate pregnancies), fetal sex (female or male), and “alcohol” (ethanol exposure at 0, 120 or 320 mg/dL) were smaller contributors to the overall composition of identified networks. 3A) Topological overlap matrix (TOM) plot for visualizing the weighted gene (protein) co-expression network, where the topological overlap considers each pair of proteins' similarity in relation to all other proteins in the network. Modules are defined by hierarchical clustering, visualized by dendrograms and module color notation. Each row or column corresponds to a single protein, where lighter red denotes low topological overlap and darker red denotes high topological overlap. 3B) Multidimensional scaling (MDS) plot showing high gene overlap and similarity across brown, yellow, blue, and green modules, while the turquoise module, containing a majority of proteins, did not exhibit overlap or similarity to other modules. 3C) Heatmap of WGCNA module significance (MS) correlations with sample traits. In the rows, modules are shown named by their corresponding colors and across the columns are the sample traits of interest. Numbers in the table correspond to the correlation coefficients between the module eigengene (ME) and the specific trait, with p-value in parentheses. The degree of correlation is illustrated with the color legend; the more intense the box color, the more positively (red) or negatively (blue) correlated is the module with the trait. The results of this analysis show that location (cell vs. EV) is the most important determinant of module identity, with the turquoise module identifying cells, and the yellow, brown, blue and green modules identifying EVs.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Derivative Assay, Expressing

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Lysosome Subcellular Located Proteins in EVs and Cells 7A) Volcano plot depicting relative expression of lysosome-localized proteins (based on UniProt Knowledgebase), with the expression of individual proteins z-score transformed across samples. 7B) Graph showing changes in average protein content as represented by the average z-score calculated for each sex and sample type (EV or cell). Pairwise analysis showed that, contrary to predictions, female fetal NSC-derived EV's expressed equivalent levels of lysosome-localized proteins compared to parental NSCs, whereas male NSC-derived EVs contained a smaller quantity of lysosome-localized proteins compared to parental NSCs. ∗∗∗ p < 0.001; ns, not significant; n = 9 for each group. 7C) Top EV enriched lysosome-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples. 7D) Top cell enriched lysosome-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing, Transformation Assay, Derivative Assay

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Endosome Subcellular Located Proteins in EVs and Cells 8A) Volcano plot depicting relative expression of endosome-localized proteins (based on UniProt Knowledgebase), with the expression of individual proteins z-score transformed across samples. 8B) Graph showing changes in average protein content as represented by the average z-score calculated for each sex and sample type (EV or cell). Pairwise analysis showed that, contrary to predictions, male fetal NSC-derived EV's expressed equivalent levels of endosome-localized proteins compared to parental NSCs, whereas female NSC-derived EVs exhibited a trend (#, p = 0.06) towards a larger quantity of endosome-localized proteins compared to parental NSCs; n = 9 for each group. 8C) Top EV enriched endosome-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples. 8D) Top cell enriched endosome-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing, Transformation Assay, Derivative Assay

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Membrane Subcellular Located Proteins in EVs and Cells 9A) Volcano plot depicting relative expression of membrane-localized proteins (based on UniProt Knowledgebase), with the expression of individual proteins z-score transformed across samples. 9B) Graph showing changes in average protein content as represented by the average z-score calculated for each sex and sample type (EV or cell). Pairwise analysis showed that, contrary to predictions, male fetal NSC-derived EV's expressed equivalent levels of membrane-localized proteins compared to parental NSCs, whereas female NSC-derived EVs exhibited a significantly larger (∗, p < 0.056) quantity of membrane-localized proteins compared to parental NSCs; n = 9 for each group; ns, not significant. 9C) Top EV enriched membrane-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples. 9D) Top cell enriched membrane-localized proteins with nonoverlapping 95% confidence estimates between EV and cell samples.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Membrane, Expressing, Transformation Assay, Derivative Assay

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Ethanol Treatment Increases the Complexity of Correlated Protein Networks in EVs. 11A) Topological overlap matrix plots depict weighted gene/protein co-expression networks in EVs (upper panel) or parental cell samples (lower panel), separated by ethanol treatment condition, 0 mg/dL, left panels; 120 mg/dL, center panels; 320 mg/dL, right panels. Modules were defined by hierarchical clustering as depicted by dendrograms and designated by a unique color identity. 11B) Bar graph depicts number of identified modules for EV and cell samples, as a function of ethanol exposure dose. Chi-squared tests determined that there was an ethanol dose-related increase in the number of modules in EVs relative to parental NSCs. ∗∗∗∗, p < 0.0001; #, p = 0.072, trending towards significance.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Effect of exposing NSCs to a Dose Range of Ethanol on the Proteome of Secreted EVs and Cells Scatter plots of effect size (Hedges' g) vs. normalized protein expression level, document that statistical significance ( p < 0.05) is independent of protein expression, but generally associated with increased effect size. 12A,B) Relationship between effect size and statistical significance in EVs from 120 mg/dL-treated NSCs (12A) or 320 mg/dL-treated NSCs (12B) relative to control (0 mg/dL) NSCs. These analyses show that more differentially regulated EV proteins also exhibited a positive effect size, indicative of upregulation in EVs. 12C,D) Relationship between effect size and statistical significance in parental NSCs treated with 120 mg/dL (12C) or 320 mg/dL (12D) relative to control (0 mg/dL) NSCs. In contrast to EVs, these analyses show that more differentially regulated parental NSC proteins also exhibited a negative effect size, indicative of down-regulation. Horizontal blue-dotted line represents the average protein expression level in EV (12A,B) or parental cell (12C,D) samples. Vertical purple-dotted lines denote effect size values of -0.8–0.4, -0.2, +0.2, +0.4, +0.8. Positive effect size signifies increased expression of a protein, and negative effect size signifies decreased expression of a protein, in treatment group vs. control group. Each data point represents a protein, with red color for significant p-value; n = 6 samples per group; paired t -test, p < 0.05. 12E,F) Volcano plots of the relationship between effect size due to treatment and p-value. Each data point represents protein enrichment in EVs relative to parental cells (EV/Cell) between moderate ethanol-treated (120 mg/dL, 12E) or heavy ethanol-treated (320 mg/dL, 12F) groups compared to the control group (0 mg/dL). These data show that proteins that were significantly enriched in EVs due to ethanol exposure were also depleted in parental NSCs. Blue triangles above the horizontal dotted line represent proteins for which the effect size had a non-zero containing 95% confidence estimate that were also significantly affected by ethanol exposure by paired t-test. Green points below the horizontal dotted line represent proteins for which the effect size had a non-zero containing 95% confidence estimate but were not significantly affected by ethanol exposure by paired t-test. Red points above the horizontal dotted line represents proteins for which the effect size had a zero containing 95% confidence estimate but were significantly affected by ethanol exposure by paired t-test; n = 6 samples per group; paired t -test, p < 0.05.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing, Control, Protein Enrichment

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: ScRNAseq of GD 14.5 Murine Developing Cortex Shows that mRNAs for Ethanol-sensitive EV-Enriched Proteins are Abundant in Ventricular Zone Cell Lineages. 14A) tSNE plot of clusters identified as part of VZ, SVZ, or TPC lineages. Data extracted from NCBI/GEO (GSE158747) ( 12 ). 14B) tSNE plot classifying VZ, SVZ, TPC lineages. 14C,D) Composite mRNA transcript expression for proteins significantly enriched in EVs, with Hedges' g > +0.4 for EVs obtained from 120 mg/dL (14C) and 320 mg/dL (14D)-treated NSCs. 14E,F) The application of a threshold cutoff of >log 2 4 for composite transcript expression shows that in vivo , neural progenitor cells of the VZ are the principal contributors of proteins that are significantly enriched in EVs following exposure of parental NSCs to 120 mg/dL (14E) or 320 mg/dL (14F) of ethanol.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Expressing, In Vivo

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Fluorescent-Labeled EVs from Donor NSCs are Sequestered by Recipient NSCs 15A) Confocal photomicrograph showing the presence of MemBrite-labeled EVs (red fluorescence) purified from donor NSCs within the cytoplasm of naïve NSCs counterstained with DAPI (blue) to visualize nuclei. 15B) Flow cytometric analysis shows that compared to unstained cells, MemBrite readily stains NSCs. Purified EVs stained with MemBrite are also readily taken up by naïve recipient cells, whereas no stain was observed in cells exposed to purified culture medium with MemBrite dye that underwent the same labeling and filtration process as isolated EVs; n = 6 to 13 samples per group; Kruskal-Wallis test for nonparametric one-way ANOVA, p < 0.0001; Dunn's multiple comparisons post-hoc test, ∗ p < 0.05.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Labeling, Fluorescence, Purification, Staining, Filtration, Isolation

Journal: Heliyon

Article Title: Dose-related shifts in proteome and function of extracellular vesicles secreted by fetal neural stem cells following chronic alcohol exposure

doi: 10.1016/j.heliyon.2022.e11348

Figure Lengend Snippet: Functional Analysis of the Effects of Purified Control EVs, and EVs Purified from Ethanol-treated NSCs on the Behavior of Naïve Recipient NSCs. Following EV administration, oxidative metabolism was measured by the alamarBlue assay (16A), glycolysis was measured by the Lactate-Glo assay (16B), and apoptosis was measured by the activation of Caspase3/7 (16C) in naïve recipient NSCs; n = 5 to 23 samples per group; repeated-measures two-way ANOVA with Geisser-Greenhouse correction; Tukey's or Dunnett's multiple comparisons post-hoc test, ∗ p < 0.05, # p < 0.08. 16D,E,F) Quantification in donor, ethanol-treated and control NSCs, of oxidative metabolic activity (16D), glycolysis activity (16E), and Caspase 3/7 apoptosis activity (16F) following moderate (120 mg/dL) or high (320 mg/dL) ethanol exposure in NSCs; n = 5 to 23 samples per group; repeated-measures two-way ANOVA with Geisser-Greenhouse correction; Tukey's multiple comparisons post-hoc test, ∗ p < 0.05. 16G) Flow cytometry analysis of the proportion of cells in S-phase of the cell cycle, following addition of EVs derived from ethanol-treated or control NSCs, to naïve recipient NSCs; n = 19 samples per group; repeated-measures two-way ANOVA with Geisser-Greenhouse correction; Dunnett's multiple comparisons post-hoc test, ∗ p < 0.05, # p < 0.08. 16H) Representative flow cytometry image.

Article Snippet: Inset depicts Nanosight image of EVs derived from NSCs.

Techniques: Functional Assay, Purification, Control, Alamar Blue Assay, Glo Assay, Activation Assay, Activity Assay, Flow Cytometry, Derivative Assay