Enantioselective Total Synthesis of CannabinoidsA Route for Analogue Development

A practical synthetic approach to Δ⁹-tetrahydrocannabinol (<b>1</b>) and cannabidiol (<b>2</b>) that provides scalable access to these natural products and should enable the generation of novel synthetic analogues is reported

Demographics of the entire CARES Cohort as well as the subset of patients that have been included in the CARES longitudinal arm and those that were used for confirmation by subcloning and sequencing.

<p>The values indicate the median and 95% confidence intervals. *Due to the longitudinal and archival nature of this dataset, the limit of detection with respect to viral load has changed but has ranged from <100 to <20.</p

The mutation rate of HIV varies over time as determined by BEAST analysis.

<p>BEAST trees from three representative patients (A0001, A0041 and A0095) are shown for both the LTR <b>(Top)</b> and <i>tat</i> exon 1 <b>(Middle)</b> regions The width of the branch indicates the rate of mutation across the branch and the branch-length represents the time since divergence. The purple nodes indicate PCR Reads, the red nodes 4.4 Kb Fragment Reads, and the green nodes indicate Clone Reads. <b>(Bottom)</b> The time-series of each patient was synchronized such that the estimated time of infection for all samples have been shifted to 0 years. The red line shows the average mutation rate of <i>tat</i> exon 1 and the red shadow shows the 95% confidence interval of the estimate. The blue line shows the average mutation rate of the LTR and the blue shadow shows the 95% confidence interval of the estimate.</p

The calculated effect sizes of fixed effects for the patients shown in Fig 3.

<p>The calculated effect sizes of fixed effects for the patients shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155382#pone.0155382.g003" target="_blank">Fig 3</a>.</p

Direct sequencing of PCR products generated directly from genomic DNA very often returns the predominant genotype.

<p><b>(A)</b> (Left) A histogram of the number of QS observed in each of the 31 samples with at least 10 Clone Reads. <b>(B)</b> A bar plot showing the fraction of the total observed QS for each unique Clone Read. Each horizontal bar represents one of the 31 samples with the width of the bar indicating the QS fraction. Each color in the bar indicates unique QS. Darker bars denote the QS that match the PCR Read.</p

Proportions of activated (CD38+HLA-DR+) and cycling (Ki67+) CD4+ and CD8+ T cells decreased following initiation of ART.

<div><p>Among both A) CD4+ and B) CD8+ T cell populations, initiation of raltegravir plus emtricitabine/tenofovir resulted in a significant decrease from baseline in the proportion of activated cells by 2 days and 7 days (p= 0.05 and 0.015, respectively). By week 48, the proportions of activated CD4+ and CD8+ T cells in these patients did not approach the proportions measured in healthy controls. Also among both C) CD4+ and D) CD8+ T cell populations, initiation of ART significantly decreased the proportions of Ki67+ cells by week 8 and by day 7 respectively (p<0.001 and p=0.028). E) Naïve CD4+ T cells (CD4+,CD45RA+, CCR7+) and F) CM CD4+ T cells (CD4+,CD45RA-, CCR7+) that express Ki67 were significantly reduced from baseline by week 8 (p=0.005 and p=0.001, respectively). In all four T cell subsets, proportions of Ki67+ cells did not approach the proportions seen in healthy controls by week 48 of the study. Symbols used in the figure:</p> <p>N = Normal controls (in blue).</p> <p>0 = Baseline.</p> <p>D = Day (The two tick-marks between “0” and “D14” are Day 2 and Day 7).</p> <p>W = Week.</p> <p>* = Change from baseline significantly different from 0 (Wilcoxon signed rank p ≤0.05) .</p> <p>x = Significant difference from the normal controls (Wilcoxon rank sum p ≤0.05).</p> <p>- Horizontal bars represent 25^th (Q1), 50^th (Median), and 75^th percentiles.</p> <p>… Dotted line (in red) connects the medians over time.</p></div

Markers of inflammation and coagulation are reduced following initiation of ART.

<div><p>Plasma samples were thawed and levels of A) interleukin-6 (IL-6) B) tumor necrosis factor receptor type 1 (TNFr1) C) D-dimers D) Lipopolysaccharide (LPS) and E) CD14 (sCD14) were measured. Initiation of ART resulted in significant decreases from baseline in plasma levels of IL-6 and TNFr1 by week 4 (p=0.002 and p=0.038) D-dimer levels were significantly reduced by day 7 (p=0.031). Levels of sCD14 were significantly reduced by day 2 following initiation of therapy (p<0.001). LPS levels were significantly reduced from baseline 24 weeks after initiation of ART (p<0.001). None of these markers, except for IL-6, consistently reached the levels seen in healthy controls by the end of the study. Symbols used in the figure: </p> <p>N = Normal controls (in blue).</p> <p>0 = Baseline.</p> <p>D = Day (The two tick-marks between “0” and “D14” are Day 2 and Day 7).</p> <p>W = Week.</p> <p>* = Change from baseline significantly different from 0 (Wilcoxon signed rank p ≤0.05) .</p> <p>x = Significant difference from the normal controls (Wilcoxon rank sum p ≤0.05).</p> <p>- Horizontal bars represent 25^th (Q1), 50^th (Median), and 75^th percentiles.</p> <p>… Dotted line (in red) connects the medians over time.</p></div

Bak silencing markedly reduces the sensitivity of T cells in chronic HIV-1 infection to CD95/Fas-induced apoptosis.

<p>(A) Efficiency of siRNA uptake in T cells transfected with a pool of siRNAs and siGlo fluorescent oligonucleotides (to monitor transfection) compared to an electroporated negative control (no RNA or siGlo). Silencing of (B) Bak and (C) Bax (off-target control) by siRNA sequences targeting Bak in primary PBMC from a representative HIV+ patient measured by quantitative PCR. Expression (mean, SEM) normalized to 18S rRNA is presented as fold change relative to non-targeting control siRNA. (D) CD95/Fas-specific apoptosis of CD4+ T cells and CD8+ T cells in chronic HIV-1 infection following transfection with a pool of siRNAs directed against Bak, relative to a pool of non-targeting siRNAs (negative control). (E) Percent inhibition of CD95/Fas-specific cell death in T cells from chronically HIV-1-infected individuals following transfection with a Bak siRNA pool. Results were obtained from 3 independent experiments with 3 different HIV-1-infected patients.</p

Bak expression is increased and correlates with CD4+ T cell counts and CD95-induced apoptosis in HIV-1-infected individuals.

<p>(A) Mean fluorescence intensity (MFI) of Bak, Bax and Bim expression <i>ex vivo</i> shown in CD4+ T cells and CD8+ T cells from HIV-1+ patients and healthy controls. Each dot depicts a donor, the lines indicate 10% and 90% and the boxes depict 25%, median and 75% quantiles. The P values were calculated by using the nonparametric Wilcoxon signed rank test for unpaired samples. (B) Spearman's rho correlation shown between <i>ex vivo</i> Bak (n = 25), Bax (n = 24) and Bim (n = 19) expression in CD4+ T cells and CD4+ T counts in HIV-1-infected individuals. (C) Spearman's rho or Pearson's r correlations shown between <i>ex vivo</i> Bak expression and the percentage of spontaneous apoptosis, AICD and CD95/Fas-mediate apoptosis in CD4+ and CD8+ T cells from HIV-1-infected individuals (n = 10).</p

Initiation of antiretroviral therapy (ART) results in an increase in the number of CD4+T cells and a decrease in the number of CD8+ T cells.

<div><p>Absolute CD4+ and CD8+ T cell counts were obtained in real time on fresh whole blood samples. Lymphocytes were identified by flow cytometry based on size and granularity; T cell subsets were identified by positive expression of CD4 or CD8. The numbers of circulating A) CD4+ and B) CD8+ T cells within this HIV-1 infected patient population changed significantly from baseline by day 2 (p<0.001 and p<0.002, respectively), but did not reach levels seen in healthy controls within 48 weeks of ART treatment. C) naïve (CD45RA+ CCR7+) and D) central memory (CM, CD45RA-CCR7+) CD4+ T cell numbers increased significantly by day 2 (p<0.001) and day 7 (p=0.001) respectively. Symbols used in the figure:</p> <p>N = Normal controls (in blue).</p> <p>0 = Baseline.</p> <p>D = Day (The two tick-marks between “0” and “D14” are Day 2 and Day 7).</p> <p>W = Week.</p> <p>* = Change from baseline significantly different from 0 (Wilcoxon signed rank p ≤0.05) .</p> <p>x = Significant difference from the normal controls (Wilcoxon rank sum p ≤0.05).</p> <p>- Horizontal bars represent 25^th (Q1), 50^th (Median), and 75^th percentiles.</p> <p>… Dotted line (in red) connects the medians over time.</p></div