Venn Diagrams illustrating the distribution of significantly altered transcripts in groups defined by global expression profiles (A) or by drugs of abuse (B).

<p> <i>A.</i> Eighty-nine transcripts were regulated in all three groups defined by global expression profiles. All of these were regulated in the same direction (increased or decreased) for Group I (DA1–18) and Group II (DA19–34), and in the opposite direction for Group III (DA35–42) cases. In all, 201/202 transcripts shared between Group I and Group II were regulated in the same direction. For Group III, 91/115 transcripts shared with Group I and 79/81 transcripts shared with Group II were regulated in the opposite direction. These data highlight the similarities of Group I and Group II, and the marked differences in Group III cases. Each cluster of three arrows indicates the direction of change in Groups I, II and III, respectively. Increased expression is indicated by ↑, a decrease by ↓, while → indicates no significant change. <i>B.</i> Cases with a drug abuse history and positive cocaine, phencyclidine or cannabinoid toxicology in blood, brain or urine were grouped into COC+, PCP+ and THC+ groups, respectively. While there were significant transcriptional differences between these groups, a total of 160 transcripts (≈2% of all transcripts) were shared for the three groups. Note that a much smaller number of transcripts were identified for the THC+ group (264 increased, 424 decreased) than for the COC+ (982 increased, 699 decreased) and PCP+ (911 increased, 1021 decreased) groups. Increased expression is indicated by ↑, decreased expression by ↓.</p

Hierarchical clustering identified three main groups of drug abuse cases.

<p>Hierarchical clustering of individual transcriptional profiles from comparisons of drug abuse cases and their individual four best-matched controls identified three main groups of drug abusers: Group I (DA1–18), Group II (DA19–34) and Group III (DA35–42). A summary of toxicology and drug abuse history for each case in the clustering dendrogram indicated cocaine use in a majority of cases, while presence of alcohol in Group I, and opioids and phencyclidine in Group II might underlie differences in Group I and II individuals. Group III cases differed markedly from other cases, which may be related to the absence or low levels of abused drug in most cases, a history of alcohol dependence, or to underlying medical conditions. Insufficient specimen for quantitative analysis of a positive hair test screening is indicated by a parenthesis around the substance name, units are ng/mg, except for cTHC (pg/mg). Abbreviations: 6AM – 6-acetyl morphine, AEME – anhydroecgonine methyl ester, BE - benzoylecgonine, CE – cocaethylene, COC – cocaine, COD - codeine, cTHC – 11-nor-9-carboxy-tetrahydrocannabinol, EEE – ecgonine ethyl ester, EME – ecgonine methyl ester, EtOH – alcohol, g% - g/dL, MDMA – N-Methyl-3,4-methylenedioxyamphetamine (Ecstasy), MOR – morphine, MTD – methadone, N/A – not available, OXYC – oxycodone, PCP – phencyclidine, THC – delta-9-tetrahydrocannabinol.</p

Validation of microarray data by quantitative PCR (QPCR).

<p>Three representatives of consistently changed functional groups (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000114#pone-0000114-t003" target="_blank">Table 3</a>) were examined by QPCR: calmodulin 2 (CALM2, blue bars), apolipoprotein 2 (APOL2, green bars), and semaphorin 3B (SEMA3B, orange bars). Numbers on the x-axis represents either the microarray-derived fold change (FC, lighter blue, green or orange bars) for each drug abuse case compared to the four best-matched control cases or the QPCR ratio (QPCR, darker blue, green or orange bars), while numbers on the y-axis represent the drug abuse case examined. QPCR validated 32/38 (84%) of the microarray data, which was performed using separate sets of brain dissections and RNA extractions from each drug abuse case and the four individual best-matched controls.</p

Demographic and sample parameters of successful culture in scalp (n = 146).

<p>OR = odds ratio, AA = African-American, W = White, PMI = postmortem interval, BMI = body mass index, F = female, M = male, VA = Virginia, DC = District of Columbia; Tox = toxicology testing in blood or vitreous humor; * = p<0.05.</p

Odds ratio of culture success: dura vs. scalp matched pairs (n = 53).

<p>No = not successful, Yes = successful.</p

Percentage of successful growth in dura (n = 53) vs. scalp (n = 146) samples.

<p>Sample of 146 scalp and 53 dural specimens. The odds of successful culture in dura were nearly 2-fold compared to scalp.</p

Fibroblast characterization: FSP-1 protein expression by immunofluorescence staining and cell proliferation assay in dura and scalp. A.

<p>The morphology of postmortem fibroblast cells generated from (a) dura and (b) scalp. Cultured cells from both sources macroscopically looked similar to what is seen in living skin fibroblast cells, with more enriched cytoplasm and spindle-shaped nuclei under phase-contrast microscopy. Cells from (c) dura and (d) scalp express cytoplasmic Fibroblast Specific Protein-1 (FSP-1) (green). Original scale bars = 35 µm. <b>B.</b> Results from cell proliferation assay in 8 fibroblast cell lines (dura and scalp from 4 individuals) in five different densities. Cell viability was determined in 24 hrs and 48 hrs by WST-8 assay. Values are the mean of results from six wells. Bars ± SE. Scalp fibroblast cell lines grew 1.27-fold faster in the same period than dura fibroblast cells. <b>C.</b> Differences in cell proliferation between scalp and dura by one-way ANOVA; scalp cell growth was significantly more rapid than dura cell growth at 24 hr and 48 hr intervals [F (1, 46) = 12.94, p<0.008].</p

iPSCs generated from one dura fibroblast line express pluripotency markers and differentiate to neuronal fates.

<p>Undifferentiated iPSCs express pluripotency markers NANOG (A) and SOX2 (B). Upon neural differentiation, these cells express neuroectoderm marker SOX1 (C). Temporal gene expression analysis also shows a decrease in pluripotency marker OCT4 (POU5F1) and an increase in <i>PAX6</i> expression by quantitative RT-PCR (D). iPSC-derived neurons express βIII-tubulin and MAP2 (E–G).</p