Age and double strand breaks repair (DSBR) rate.

<p>Simple linear regression of double strand breaks repair (DSBR) rate shown in response to age (years).</p

γ-H2AX yields repair kinetics based on demographic groups.

<p>(a) Experimental data and model fit (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121083#pone.0121083.e001" target="_blank">Equation 1</a>) of γ-H2AX foci repair kinetics at 0.5 h, 2 h, 4 h, 7 h and 24 h post ex vivo gamma irradiation based on (A) age (B) alcohol use (C) gender (D) ethnicity (E) and (F) racial groups. The data obtained is plotted as points with error bars. The predictions from the model are depicted with correspondingly colored dotted lines. Error bars are ±SEM.</p

Age, ethnicity and γH2AX endogenous levels.

<p>Effect of age on variation in γH2AX endogenous levels on ethnicity is shown with simple linear regression.</p

Donor γ-H2AX yields repair kinetics.

<p>Experimental data and model fit (from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0121083#pone.0121083.e001" target="_blank">Equation 1</a>) of γ-H2AX yields repair kinetics pooled from 94 donors exposed <i>ex vivo</i> to 4 Gy gamma radiation at 0.5 h, 2 h, 4 h, 7 h and 24 h post irradiation. Error bars represent ± SEM.</p

Whole cell lipidome of control and LB-rich RBL2H3.

<p>RBL2H3 were grown for 6d with insulin-FDI. Individual major lipid species were separated by high performance liquid chromatography (HPLC) and fatty acid methyl esters (FAME) from each class were produced and subsequently analyzed by GC/MS. <b>A.</b> The total fold change in nanomolar amounts of each fatty acid within each class of lipids are presented in a heatmap using RColorBrewer and gplots in R. Negative fold change moves towards purple whereas positive fold change moves toward green. Trace lines are used to reinforce change within the groups and are relative to a dashed median line. The density of change is tracked within the scale bar to the left. <b>B.</b> Change in absolute TAG and FFA levels summed between untreated and insulin-treated cell samples. <b>C</b>. The average fold change of fatty acids between the two conditions is bar plotted and organized by the degree of saturation (saturated, mono-unsaturated (MUFA), and poly-unsaturated (PUFA)) of the fatty acids. The legend on the right side indicates the scale in fold change from -3.5 to 6. <b>D.</b> Individual FA in the AA biosynthetic pathway were quantified according to their respective major lipid class. Fold change was calculated based on response to insulin-FDI in treated compared to control mast cells (18:2n6, linoleic acid; 18:3n6, linolenic acid; 20:3n6, di-homo-gamma-linolenic acid; 20:4n6, arachidonic acid). <b>E.</b> Experiment as in <b>D</b>, with quantification of individual FA directly involved in the AA biosynthesis pathway quantified by lipid class in terms of absolute concentration (nmol of lipid per billion cells). <i>Cholesterol Ester (CE)</i>, <i>Cardiolipin (CL)</i>, <i>Triacylglycerol (TAG)</i>, <i>Diacylglycerol (DAG)</i>, <i>Free Fatty Acid (FFA)</i>, <i>Phosphatidylserine (PS)</i>, <i>Phosphatidylcholine (PC)</i>, <i>Phosphatidylethanolamide (PE)</i>, <i>Lysophoshatidylcholine (LYPC)</i>.</p

Chronic Insulin Exposure Induces ER Stress and Lipid Body Accumulation in Mast Cells at the Expense of Their Secretory Degranulation Response

<div><p>Lipid bodies (LB) are reservoirs of precursors to inflammatory lipid mediators in immunocytes, including mast cells. LB numbers are dynamic, increasing dramatically under conditions of immunological challenge. We have previously shown <i>in vitro</i> that insulin-influenced lipogenic pathways induce LB biogenesis in mast cells, with their numbers attaining steatosis-like levels. Here, we demonstrate that <i>in vivo</i> hyperinsulinemia resulting from high fat diet is associated with LB accumulation in murine mast cells and basophils. We characterize the lipidome of purified insulin-induced LB, and the shifts in the whole cell lipid landscape in LB that are associated with their accumulation, in both model (RBL2H3) and primary mast cells. Lipidomic analysis suggests a gain of function associated with LB accumulation, in terms of elevated levels of eicosanoid precursors that translate to enhanced antigen-induced LTC4 release. Loss-of-function in terms of a suppressed degranulation response was also associated with LB accumulation, as were ER reprogramming and ER stress, analogous to observations in the obese hepatocyte and adipocyte. Taken together, these data suggest that chronic insulin elevation drives mast cell LB enrichment <i>in vitro</i> and <i>in vivo</i>, with associated effects on the cellular lipidome, ER status and pro-inflammatory responses.</p></div

Whole cell lipidome of control and LB-rich RBL2H3.

<p>RBL2H3 were grown for 6d with insulin-FDI. Individual major lipid species were separated by high performance liquid chromatography (HPLC) and fatty acid methyl esters (FAME) from each class were produced and subsequently analyzed by GC/MS. <b>A.</b> The total fold change in nanomolar amounts of each fatty acid within each class of lipids are presented in a heatmap using RColorBrewer and gplots in R. Negative fold change moves towards purple whereas positive fold change moves toward green. Trace lines are used to reinforce change within the groups and are relative to a dashed median line. The density of change is tracked within the scale bar to the left. <b>B.</b> Change in absolute TAG and FFA levels summed between untreated and insulin-treated cell samples. <b>C</b>. The average fold change of fatty acids between the two conditions is bar plotted and organized by the degree of saturation (saturated, mono-unsaturated (MUFA), and poly-unsaturated (PUFA)) of the fatty acids. The legend on the right side indicates the scale in fold change from -3.5 to 6. <b>D.</b> Individual FA in the AA biosynthetic pathway were quantified according to their respective major lipid class. Fold change was calculated based on response to insulin-FDI in treated compared to control mast cells (18:2n6, linoleic acid; 18:3n6, linolenic acid; 20:3n6, di-homo-gamma-linolenic acid; 20:4n6, arachidonic acid). <b>E.</b> Experiment as in <b>D</b>, with quantification of individual FA directly involved in the AA biosynthesis pathway quantified by lipid class in terms of absolute concentration (nmol of lipid per billion cells). <i>Cholesterol Ester (CE)</i>, <i>Cardiolipin (CL)</i>, <i>Triacylglycerol (TAG)</i>, <i>Diacylglycerol (DAG)</i>, <i>Free Fatty Acid (FFA)</i>, <i>Phosphatidylserine (PS)</i>, <i>Phosphatidylcholine (PC)</i>, <i>Phosphatidylethanolamide (PE)</i>, <i>Lysophoshatidylcholine (LYPC)</i>.</p

Image1_Identification of minimum essential therapeutic mixtures from cannabis plant extracts by screening in cell and animal models of Parkinson’s disease.TIF

Medicinal cannabis has shown promise for the symptomatic treatment of Parkinson’s disease (PD), but patient exposure to whole plant mixtures may be undesirable due to concerns around safety, consistency, regulatory issues, and psychoactivity. Identification of a subset of components responsible for the potential therapeutic effects within cannabis represents a direct path forward for the generation of anti-PD drugs. Using an in silico database, literature reviews, and cell based assays, GB Sciences previously identified and patented a subset of five cannabinoids and five terpenes that could potentially recapitulate the anti-PD attributes of cannabis. While this work represents a critical step towards harnessing the anti-PD capabilities of cannabis, polypharmaceutical drugs of this complexity may not be feasible as therapeutics. In this paper, we utilize a reductionist approach to identify minimal essential mixtures (MEMs) of these components that are amenable to pharmacological formulation. In the first phase, cell-based models revealed that the cannabinoids had the most significant positive effects on neuroprotection and dopamine secretion. We then evaluated the ability of combinations of these cannabinoids to ameliorate a 6-hydroxydopmamine (OHDA)-induced change in locomotion in larval zebrafish, which has become a well-established PD disease model. Equimolar mixtures that each contained three cannabinoids were able to significantly reverse the OHDA mediated changes in locomotion and other advanced metrics of behavior. Additional screening of sixty-three variations of the original cannabinoid mixtures identified five highly efficacious mixtures that outperformed the original equimolar cannabinoid MEMs and represent the most attractive candidates for therapeutic development. This work highlights the strength of the reductionist approach for the development of ratio-controlled, cannabis mixture-based therapeutics for the treatment of Parkinson’s disease.</p

ER distension and lipid biogenesis in insulin-exposed RBL2H3.

<p><b>A. Electron micrographs of control and insulin-FDI exposed RBL2H3 showing normal (left) and distended (right) ER.</b> Images are digitally zoomed from 5000x original plates. <b>B. Area analysis of ER by electron and fluorescence microscopy</b>. Cytoplasmic (n of 20 per condition) ROI were drawn on either micrographs (Image J) or confocal images of cells stained with ER-Tracker dye (NIS Elements). Data are expressed as percentage area of ROI occupied by ER. <b>C. Confirmation of ER enrichment in purified ER/microsomal fractions.</b> ER/microsomal fractions were prepared by ultracentrifugation as described in Methods and Western blotted for enrichment in the ER-resident chaperone Calnexin. Relative band intensities are shown on each panel (Image J). <b>D. Comparison of protein and lipid levels in ER/microsomal fractions prepared from control and Insulin-FDI treated RBL2H3</b>. Total protein was assessed by BCA analysis, and total lipid content was assessed by ORO absorbance assay after Bligh-Dyer extraction. <b>E. Characterization of ER Fatty acids</b>. RBL2H3 were grown for 6d with insulin-FDI as described. Isolated ER was analyzed by GC/MS and variations in ER lipids versus control levels were organized by fold change (values along y-axis) and abundance. <i>Green</i>, > 2 fold increase in treated over controls; <i>gray</i>, no change; <i>red</i>, decrease to < 50% of control levels. <b>F. Relative abundance of eicosatrienoic, arachidonic and linoleic acid in ER from control and Insulin-FDI treated cells</b>. <b>G. Summary of alterations in saturated (SFA), mono-unsaturated (MUFA) and poly-unsaturated (PUFA) fatty acids in ER/microsomal fractions from control and insulin FDI-treated cells</b>.</p

ER reprogramming, ER stress and autophagy in insulin-treated RBL2H3.

<p><b>A, B. Altered expression of markers of ER stress, the UPR and autophagy.</b> RBL2H3 were treated with insulin-FDI for 6d and protein lysates were prepared. Western blot analysis (antibody concentrations indicated in micrograms/ml)was performed using UPR markers anti-IRE1 alpha (0.5), anti-phospho PERK-Thr980 (0.1), anti-ATF6 (2.5) and loading control anti-GRB2 (0.05)(A) and autophagy markers (B) anti-ATG3 (0.5), anti-ATG12 (0.5), anti-ATG7 (0.5), anti-Beclin (0.5), anti-LC3A (0.1), and anti-LC3B (0.5) with anti-Grb2 as a loading control. <b>C-E.</b> Immunofluorescent identification and quantification of autophagy positive mast cells. Three markers of autophagy (Beclin-1, LC3B and ATG7) were used to quantify the percent of cells staining positively for autophagy (<b>C</b>). <b>D, E</b>. Quantification of autophagy marker immunostaining. Counting was performed in a sample-blinded fashion and expressed as % of 200 counted cells (<b>D</b>) and mean of the number of immunodecorated structures per cell (<b>E</b>).</p