Subdivision of sleep states by velocity.

<p>States of sleep were subdivided into high velocity states (i.e. transitions and fluctuations) (<b>a</b>), and slow states (i.e. stable clusters) (<b>b</b>), by a heuristic velocity limit. Points are colour coded according to velocity (right side: colour bar legend: arbitrary velocity units). Comparison of the manual classification (<b>c</b>) with automatic probability based classification (<b>d</b>) for a whole night data set for one representative subject is shown. Colour coding: WAKE (red), NREM stage 1 (yellow), stage 2 (green), stage 3 (blue), and REM sleep (magenta).</p

Conserved topography of the sleep state space.

<p>(<b>a</b>) Summary scatter plot of all sleep states for 14 subjects mapped in a 2-dimensional state space. Each 5<b> </b>s EEG epoch (raw data) is represented by 2 different frequency ratios plotted on log/log axes. Ratio1 = (8.6 to 19.3 Hz)/(1.0 to 10.9 Hz), Ratio2 = (11.5 to 20.3 Hz)/(17.9 to 31.5 Hz). Colour coding of the clusters is based on expert scoring for WAKE (red), NREM stage 1 (yellow), stage 2 (green), stage 3 (blue), and REM sleep (magenta). Projections of the 2d-probability density distributions are plotted for NREM stage 2 and stage 3 (top edge, ratio1) and WAKE and REM sleep (right edge, ratio2). For better visibility, the figure shows 10% of all data after applying a running window average (6 point-Hann Window) on the raw data to filter for short-term fluctuations. (<b>b</b>) Individual sleep trajectories are shown for each subject separately (panel 1–14) and cumulated for all individuals (panel 15, bottom right). Sleep trajectories are smoothed (50 point-Hann Window) for better differentiation of stable (clusters) and transitional sleep states (trajectories). Colour coding is as described in (a).</p

Laterality Analysis of REM sleep.

<p>(<b>a</b>) Time series data of relative laterality of velocity in corresponding central electrodes (C3 vs C4) for 100 successive REM sleep epochs for one individual. (<b>b</b>) Sample autocorrelation of the same period as shown in (a) demonstrating a stable oscillation with a period of about 40<b> </b>s. Approximate 95% confidence measures for the hypothesis of uncorrelated white noise are indicated by a dotted thin blue horizontal line (p = 0.05, N = 100). (<b>c</b>) Fourier analysis (FFT) of REM sleep autocorrelations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048660#pone-0048660-g004" target="_blank">Figure 4b</a> during one selected REM sleep bout for each subject. (<b>d</b>) Mean spectral analysis of REM sleep autocorrelation of all 14 subjects.</p

Demographic data.

<p>Demographic data.</p

MTHFR variants in smokers and non-smokers.

<p>Genotype frequencies concerning the two alleles MTHFR c.6777C>T and MTHFR c.1298A>C as well as the genotype frequencies resulting from the c.677C>T;c.1298A>C haplotypes.</p

Homocysteine levels and MTHFR variants.

<p>Fasting total plasma homocysteine levels in µmol/l ± one standard deviation.</p

<i>Cst7</i>/cystatin F brain expression during prion pathogenesis.

<p><b>A</b> Western blotting analysis in brain extracts of terminally sick, prion-infected mice (RML), and of mice injected with non-infectious brain homogenate (NBH), showing the amount of partially protease K (PK)-resistant prion protein (PrP) (left panel). Identical protein extracts omitting proteinase K treatment were used for Western blotting with an anti-PrP and anti-actin antibody to verify equivalent loading in each lane (right panel). ZH3 denotes brain from a C57BL/6J-<i>Prnp</i>^ZH3/ZH3 mouse as control. Each lane denotes one mouse. <b>B</b> Cystatin F levels in brain extracts from the same mice depicted in A, relative to levels in NBH-injected mice (set as 100%; ***, p<0.001, Student’s t test). <b>C</b> Cystatin F levels in brain extracts from mice injected with either non-infectious brain homogenate (NBH) or 22L prions at different time points (dpi, days post-inoculation; term, terminal prion disease, reached at approximately 150 pdi in this experiment), relative to levels in NBH-injected mice at 150 dpi (ns, p>0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001, one-way ANOVA followed by Bonferroni’s Multiple Comparison test). <b>D</b> Histological analysis of cystatin F expression in cerebella of 22L-inoculated mice at the terminal stage of the disease and the relative NBH-injected controls. Three representative mice per each group are depicted. Scale bar: 100 μm.</p

Microarray analysis identifies Cst7 as the most upregulated transcript in RML inoculated mice.

<p><b>A</b> Heatmap showing relative expression of 251 differentially expressed genes (absolute fold change ≥1.3, a confidence interval >90% and Student’s t test p value <0.05) between RML prion-inoculated mice (RML, n = 3, A to C) and control mice injected with non-infectious brain homogenates (NBH, n = 3, A to C). For each gene, relative expression is defined as the ratio between the actual gene expression of each mouse and the average gene expression from all 6 analyzed mice (the latter set as 1, black) and is reported as grades of blue (downregulation) or red (upregulation). Expression data were obtained through the employment of the perfect match mismatch model-based expression analysis algorithm of microarray data previously reported in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171923#pone.0171923.ref006" target="_blank">6</a>]. The list of differentially expressed genes is reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171923#pone.0171923.s010" target="_blank">S4 Table</a>. <b>B</b> Validation of the upregulation of 29 transcripts (including <i>Cst7</i>) in RML-inoculated mice (n = 3) compared to NBH-injected controls (n = 3). Bars (left y axis) depict mean and standard deviation of transcript fold change expression between brains of RML-inoculated mice and NBH-injected controls (after normalization to <i>Actb</i> levels) at 145 and 190 days post-inoculation (dpi), as assessed by RT-PCR. Red diamonds (right y axis) depict transcript fold change expression between brains of RML-inoculated mice and NBH-injected controls at 145 dpi as assessed by microarray.</p

Cystatin F levels in brains from patients with Creutzfeldt-Jakob disease and Alzheimer’s disease.

<p><b>A</b> Cystatin F levels in frontal cortex (FCx) and cerebellum (Cb) of patients with autopsy-confirmed definitive Creutzfeldt-Jakob disease (Def CJD) or Alzheimer’s disease (Def AD). Dots: denote individual subjects; bars: mean; error bars: standard deviation. <b>B</b> Correlation of cystatin F levels in the two regions of each patient, denoted by a dot (R² 0.73 for Def AD and 0.83 for CJD, linear regression analysis). <b>C</b> Histologic analysis of cystatin F expression in frontal cortex of the same cohort of patients. Regions with abundant pathology, including amyloid β (Aβ) plaques and neurofibrillary tangles (Tau, further magnified in the inset) in Alzheimer’s disease and spongiosis and partially protease resistant prion protein (PrP) in Creutzfeldt-Jakob disease, from representative cases are depicted. Non-demented subjects were included as control (Ctrl). Lower row, left: encephalitis, with cystatin F positive infiltrating cells (further magnified in the inset at center). Lower row, right: bone marrow, with cystatin F positive cells (further magnified in the inset). Scale bar: 100 μm (black) or 25 μm (blue).</p

Cystatin F levels in cerebrospinal fluid from patients with Creutzfeldt-Jakob diseases, Alzheimer’s disease and controls.

<p>Cystatin F levels were measured in patients with autopsy-confirmed definitive Creutzfeldt-Jakob disease (Def CJD) or Alzheimer’s disease (Def AD), clinically diagnosed Alzheimer’s disease (Clin AD) and non-demented controls (Controls). <b>A</b> Age and gender distribution (****, p<0.0001 against all other categories, one-way ANOVA followed by Bonferroni’s Multiple Comparison test). <b>B</b> Sample storage of archival cerebrospinal fluid (CSF). <b>C</b> Time interval between lumbar puncture (LP) and <i>exitus</i> for deceased patients with autopsy-confirmed diagnosis of neurodegeneration (Def AD and Def CJD; **, p<0.01, Student’s t test). <b>D</b> Cystatin F levels in CSF from all study subjects from cohort 2 (*, p<0.05, one-way ANOVA followed by Bonferroni’s Multiple Comparison test). <b>E</b> Sub-analysis of cystatin F levels in CSF from Def CJD subjects according to the PrP type[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171923#pone.0171923.ref035" target="_blank">35</a>]. <b>F</b> Sub-analysis of cystatin F levels in CSF from Def CJD subjects according to the results of p14-3-3 Western blotting analysis as performed at the time of CSF sample collection. In all panels: Dots: denote individual subjects; bars: mean; error bars: standard deviation.</p