Urine Tx-M/PGI-M ratio in compliant ADAPT participants who self-reported ASA use was analyzed by two-way ANOVA; treatment group assignment (P<0.01), but not ASA use significantly affected this ratio.

<p>Bonferroni-corrected post-hoc t-tests showed that among treatment groups, only Placebo was significantly different (P<0.05) between ASA and No ASA users.</p

Urine Tx-M and PGI-M concentrations in ADAT participants categorized by treatment adherence, group assignment, and aspirin use.

<p>Two-way ANOVAs were performed for each eicosanoid in compliant and non-compliant subjects for the three treatment groups. Group assignment was significantly (⁺P<0.0001) related to urine Tx-M and PGI-M in the compliant group, but not for the non-compliant group (^∧P>0.05). ASA use was associated with reduced urine Tx-M concentrations (^#P<0.05) in the compliant and non-compliant groups, and reduced urine PGI-M in only the compliant group (^#P<0.05). A significant interaction between group assignment and ASA use (P<0.05) was detected only for urine Tx-M concentrations in the compliant group. Bonferroni-corrected post-hoc comparisons evaluated the effects of ASA use in each of the twelve groups; only urine Tx-M in compliant Placebo and compliant celecoxib participants was significant (*P<0.0001) while all others had P>0.05. In addition, values for ASA compliant and noncompliant individuals were calculated regardless of treatment group assignment, presented in rows labeled subtotal, and compared using t-tests (⁺⁺P<0.0001, **P<0.05).</p

Male Microchimerism in the Human Female Brain

<div><p>In humans, naturally acquired microchimerism has been observed in many tissues and organs. Fetal microchimerism, however, has not been investigated in the human brain. Microchimerism of fetal as well as maternal origin has recently been reported in the mouse brain. In this study, we quantified male DNA in the human female brain as a marker for microchimerism of fetal origin (i.e. acquisition of male DNA by a woman while bearing a male fetus). Targeting the Y-chromosome-specific <em>DYS14</em> gene, we performed real-time quantitative PCR in autopsied brain from women without clinical or pathologic evidence of neurologic disease (n = 26), or women who had Alzheimer’s disease (n = 33). We report that 63% of the females (37 of 59) tested harbored male microchimerism in the brain. Male microchimerism was present in multiple brain regions. Results also suggested lower prevalence (p = 0.03) and concentration (p = 0.06) of male microchimerism in the brains of women with Alzheimer’s disease than the brains of women without neurologic disease. In conclusion, male microchimerism is frequent and widely distributed in the human female brain.</p> </div

Concentration of male Mc in female human brain regions.

<p>Autopsied brain specimens of females without any neurologic disease (open circles) or with AD (filled circles) were tested by qPCR for male DNA. Each point represents one unique brain specimen. Telencephalon consists of neocortical regions (frontal, parietal, temporal, and occipital lobes), limbic regions (hippocampus, amygdala, and cingulate gyrus), and regions of the basal ganglia (putamen, caudate, and globus pallidus). Diencephalon consists of thalamus. Rhombencephalon consists of medulla, pons, and cerebellum. Due to the greater number of data points collected for telencephalon and rhombencephalon, data for each group have been plotted side by side to better present their distributions. Such separation was not done on the data for diencephalon and spinal cord.</p

Characteristics of female subjects without neurologic disease or with Alzheimer’s disease.

<p>Characteristics of female subjects without neurologic disease or with Alzheimer’s disease.</p

Prevalence of male Mc within individual brain regions in women without neurologic disease or with Alzheimer’s disease.

*<p>P = 0.03 comparing the overall prevalence of male Mc between the two groups. Individual brain regions were not compared due to limited sample sizes.</p

A <i>Drosophila dGBA1b</i> deletion results in glucocerebrosidase deficiency.

<p>(A) Comparison of protein sequence of human GBA1 and <i>Drosophila</i> dGBA1a and dGBA1b. Gray indicates similar residues, whereas black shading indicates identical residues. (B) Genomic organization of the <i>Drosophila GBA1</i> homologs, <i>dGBA1a</i> and <i>dGBA1b</i>, and the intervening <i>CG31413</i> gene. Orange and blue boxes represent coding and non-coding sequences, respectively. Black arrows indicate direction of transcription. Red arrows designate the breakpoints of the <i>GBA1</i>^<i>ΔTT</i> deletion allele. (C) There was no significant difference in the percentage of <i>GBA1</i>^<i>ΔTT</i> homozygotes and WT controls (<i>GBA1</i>^<i>+/+</i>) that survived the embryo to 1st instar larval transition, the 3^rd instar larval to pupal stage transition, or the pupal to adult stage transition. (D) Relative glucocerebrosidase (GCase) enzyme activity from isolated heads of 14-day-old controls and <i>GBA1</i>^<i>ΔTT</i> homozygotes. (E) Relative GCase enzyme activity from bodies excluding heads of 14-day-old controls and <i>GBA1</i>^<i>ΔTT</i> homozygotes. Error bars represent standard error of the mean (s.e.m.), ns indicates p>0.05, **<i>p</i><0.005 by Student <i>t</i> test in all results shown in this figure.</p

<i>GBA1</i>^<i>ΔTT</i> homozygotes exhibit shortened lifespan and behavioral phenotypes consistent with neuronal dysfunction.

<p>(A) Kaplan-Meier survival curves of WT controls (<i>GBA1</i>^<i>+/+</i>), <i>GBA1</i>^<i>ΔTT</i> heterozygotes (<i>GBA1</i>^{<i>ΔTT/+</i>}), <i>GBA1</i>^<i>ΔTT</i> homozygotes (<i>GBA1</i>^{<i>ΔTT/ΔTT</i>}), WT controls ectopically expressing <i>Drosophila</i> WT <i>dGBA1b</i> using the <i>Actin GAL4</i> driver (<i>Actin-GAL4>UAS-GBA1b;GBA</i>^<i>+/+</i>), <i>GBA1</i>^<i>ΔTT</i> homozygotes ectopically expressing <i>Drosophila</i> WT <i>dGBA1b</i> using the <i>Actin GAL4</i> driver <i>(Act-GAL4>UAS-GBA1b;GBA1</i>^{<i>ΔTT/ΔTT</i>}), <i>GBA1</i>^<i>ΔTT</i> homozygotes ectopically expressing human WT <i>GBA1</i> using the <i>Actin GAL4</i> driver (<i>Act-GAL4>UAS-hGBA1</i>^<i>WT</i><i>;GBA1</i>^{<i>ΔTT/ΔTT</i>}), and <i>GBA1</i>^<i>ΔTT</i> homozygotes ectopically expressing human <i>GBA1</i> harboring the p.<i>N370S</i> mutation using the <i>Actin GAL4</i> driver (<i>Act-GAL4>UAS-hGBA1</i>^<i>N370S</i><i>;GBA1</i>^{<i>ΔTT/ΔTT</i>}). (B) Climbing index of 5-day-old flies of indicated genotypes as described in A. (C) Recovery time from mechanical stress (bang sensitivity) of flies of given genotypes as described in A at given adult ages. (D) Recovery time from heat stress of flies of given phenotypes as described in A at given adult ages. Error bars represent s.e.m., *<i>p</i><0.05, **<i>p</i><0.005 by Student <i>t</i> test in all results shown in this figure.</p

<i>GBA1</i>^<i>ΔTT</i> homozygotes display a memory deficit and neurodegeneration, but do not have dopaminergic neuron loss.

<p>(A) Latency time to initiate courtship in untrained 14-day-old males of indicated genotype. (B) Latency time to initiate courtship in 14-day-old males of indicated genotypes at 1 hour, 6 hours and 24 hours following training using a conditioned mating assay. Note the longer latency times in trained males (B) relative to untrained males (A). (C) Representative paraffin-embedded H&E-stained brain sections from 30-day-old controls and <i>GBA1</i>^<i>ΔTT</i> homozygotes. Yellow arrows indicate vacuoles. (D) Representative image of a projected Z-series of a control adult <i>Drosophila</i> brain stained with anti-Tyrosine Hydroxylase to label dopaminergic (DA) neurons. DA neurons within the PPL1 cluster are indicated by the circled regions. Scale bar, 200 μm. (E) Relative number of DA neurons within the PPL1 cluster of 30-day-old <i>GBA1</i>^<i>ΔTT</i> homozygotes (<i>GBA1</i>^{<i>ΔTT/ΔTT</i>}) <i>N</i> = 19, normalized to age-matched WT controls (<i>GBA1</i>^<i>+/+</i>) <i>N</i> = 21. There was no significant difference between the number of DA neurons within the PPL1 cluster per genotype by Student <i>t</i> test. Error bars represent s.e.m., ns indicates <i>p</i>>0.05, *<i>p</i><0.05, **<i>p</i><0.005 by Student <i>t</i> test in all results shown in this figure.</p

<i>GBA1</i>^{<i>ΔTT/ΔTT</i>} exhibit increased accumulation of soluble and insoluble ubiquitinated proteins.

<p>(A) Western blot using antiserum to ubiquitin (Ub) of Triton-soluble (S) and Triton–insoluble (I) protein fractions from whole 30-day-old WT controls (<i>GBA1</i>^<i>+/+</i>) and <i>GBA1</i>^<i>ΔTT</i> homozygotes (<i>GBA1</i>^{<i>ΔTT/ΔTT</i>}), using anti-β-Actin (βAct) as a loading control. The intense ubiquitin-positive band immediately below the 72kD marker and the upper βAct band likely represents arthirin, a 55kD mono-ubiquitinated form of β-Actin that is present exclusively in the indirect flight muscle [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005944#pgen.1005944.ref068" target="_blank">68</a>]. (B) Western blot probed for Ub of Triton–soluble (S) and Triton–insoluble (I) protein fractions from heads of flies of 30-day-old <i>GBA1</i>^<i>+/+</i>, <i>GBA1</i>^{<i>ΔTT/ΔTT</i>}, WT controls ectopically expressing <i>Drosophila</i> WT <i>dGBA1b</i> using the <i>Actin GAL4</i> driver (<i>Actin-GAL4>UAS-GBA1b;GBA</i>^<i>+/+</i>), and <i>GBA1</i>^<i>ΔTT</i> homozygotes ectopically expressing <i>Drosophila</i> WT <i>dGBA1b</i> using the <i>Actin GAL4</i> driver <i>(Act-GAL4>UAS-GBAb;GBA1</i>^{<i>ΔTT/ΔTT</i>}), using βAct as a loading control. (C) Densitometric quantification of Ub signal in the Triton-insoluble fraction from the heads of flies of the indicated genotypes. Levels of Ub signal per genotype were normalized to respective βAct loading controls, and these ratios were in turn normalized to the insoluble Ub level of <i>GBA1</i>^<i>+/+</i>. (D) Representative immunofluorescent staining of thoracic muscle from 30-day-old <i>GBA1</i>^<i>+/+</i> and <i>GBA1</i>^{<i>ΔTT/ΔTT</i>} flies with anti-Ub and anti-F-Actin. Scale bars, 20 μm. (E) Quantification of Ub-positive objects within 10-μm-thick <i>Z</i>-stacks of thoracic muscle of 30-day-old <i>GBA1</i>^<i>+/+</i> and <i>GBA1</i>^{<i>ΔTT/ΔTT</i>} flies. At least 9 separate Z-stacks were analyzed per genotype. Error bars represent standard deviation. (F) Representative anti-Ub immunofluorescent staining of 30-day-old whole brains from <i>GBA1</i>^<i>+/+</i> and <i>GBA1</i>^{<i>ΔTT/ΔTT</i>} flies. Scale bar, 200 μm. Arrowhead indicates punctate staining pattern, arrow indicates filamentous staining pattern. (G) Western blot using antiserum to Ref(2)P of whole 10-day-old flies of the indicated genotypes, including <i>Atg7</i> null flies <i>(Atg7</i>^{<i>d77/d77</i>}). βAct was used as a loading control. (H) Densitometric quantification of Ref(2)P signal from 10-day-old whole flies of the indicated genotypes. Levels of Ref(2)P signal per genotype were normalized to respective βAct loading controls, and these ratios were in turn normalized to the Ref(2)p level of <i>GBA1</i>^<i>+/+</i>. (I) Cathepsin D activity in whole 7-day-old <i>GBA1</i>^<i>ΔTT</i> homozygotes relative to age-matched controls. (J) Hexosaminidase activity in bodies excluding heads, and isolated heads of 7-day-old <i>GBA1</i>^<i>ΔTT</i> homozygotes relative to age-matched controls. Error bars represent s.e.m. unless indicated, ns indicates <i>p</i>>0.05, *<i>p</i><0.05, **<i>p</i><0.005 by Student <i>t</i> test in all results shown in this figure.</p