Pulsicons for All Participants.

<p>The table is organized by number of iterations: 3 for 9 participants and 4 for 5 participants. As iteration level increases, the representation appears simpler.</p><p>Pulsicons for All Participants.</p

Qualitative time-series representations for a single participant (ID6).

<p>The dotted vertical lines with lower case Roman numerals represent the same time points in the Time-Series, the Pulsicon, and the Production Graph for participant ID6. Shaded rectangles labeled with uppercase roman numerals represent the same temporal interval in the Time-Series, the Pulsicon, and the Production Graph. <b>Time-Series:</b> The 24-hour cortisol time-series. Nadirs identified in each of the four HAP iterations are connected with lines. The terminal HAP nadirs are identified with black circles. <b>Pulsicon:</b> The pulsicon for the time-series. <b>Production Graph:</b> Each rectangle represents productions: the application of rules defined in the CFL The five rectangular boxes represent the key features of the time-series. The first two rectangles (Labeled 1 and 2) show that there is a decreasing portion prior to the main portion of the cortisol time-series with the “>” in rectangle 2 (e.g., before left black circle). The main portion of the cortisol time-series is represented by the center rectangle (Labeled 3) with the production symbol (S). The production symbol (S) is a variable that can be replaced with any of the rules defined in the context free language. This section contains a hierarchy of embedded pulses. The last two rectangles (Labeled 4 and 5) from the production graph show that the cortisol time-series is rising after the main portion (rectangle labeled 3) of the cortisol time-series (e.g., after right black circle).</p

Amplitude (A) and secretion times (T) for simulated dataset*.

<p>Bolded entries were not identified by HAP Analysis.</p><p>Amplitude (A) and secretion times (T) for simulated dataset*.</p

Accumulation and dissipation rates computed from the Level 1 iteration for all participants.

<p>Accumulation and dissipation rates computed from the Level 1 iteration for all participants.</p

Multi-scale analysis of features identified with HAP from a single participant (ID6).

<p>Each analysis iteration for participant ID6 is shown with a different symbol: 1-circle, 2-square, 3-up triangle, 4-down triangle. The symbol color indicates the timing of the event with the color code in right-most vertical panel: the sleep episode (blue), the first 8 hours of wake (green), or second 8 hours of wake (white). The dashed lines connect median value between iterations. (<b>A</b>) Cortisol accumulation rates for nadirs identified at each iteration. Accumulation rates for the first iteration are analogous to secretory rates. (<b>B</b>) Cortisol dissipation rates for nadirs identified at each iteration. Dissipation rates for the first iteration are analogous to clearance rates. (<b>C</b>) The nadir intervals identified at each iteration are shown. The event intervals at the first iteration are analogous to the inter-pulse interval. (<b>D</b>) The relationship between an accumulation rate and the immediately following dissipation rate.</p

Hierarchically embedded cortisol pulses.

<p>An example adapted from experimental data (black line) with six major cortisol pulses. For a <b>Single Pulse Rise (U₁)</b> starting at time ∼3.5 hours, the single pulse peak is identified with and the starting nadir/valley is identified with a V₁. The pulse rise is defined as the rise in cortisol concentration from the nadir (V₁) to peak (P₁). The pulse rise time (Uw₁) is the time required for the cortisol concentration levels to rise from a sequence of pulses starting with the local nadir (V₁) to the peak (P₁). The <b>Hierarchical (multiple) Pulse Rise (U₂)</b> occurs as the change in concentration from the first nadir (V₂) to last nadir (P₂), which is a local maximum nadir, in the rise portion of this hierarchically organized segment. The hierarchical pulse rise time (Uw₂) is the elapsed time between V₂ and P₂ of the rising portion. The effect of the hierarchical pulses between V₂ and P₂ is an accumulation or increase in cortisol. Similarly, sequences of pulses associated with the dissipation or decrease in cortisol begin at time ∼4.8 hours.</p

Simulated 24-hour cortisol concentration time-series profiles with gamma values from Table 1.

<p>Within each panel, the left figure plots the time and amplitudes of each pulse and the right figure plots the resulting simulated dataset. The gamma value affects the clearance rate. (A) Gamma = 0.25 (B) Gamma = 0.75 (C) Gamma = 1.0 (D) Gamma = 1.5 (E) Gamma = 2.5, (F) Gamma = 5.0.</p

HAP analysis of simulated cortisol test set.

<p>(A–F) Simulated data generated from the data sets shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104087#pone-0104087-g004" target="_blank">Fig. 4</a> panels A–F. Simulated data are identified with blue markers. Peaks identified during the nadir selection algorithm are in red. Nadirs identified at a given recursive step are joined with a line, where line color represents a different recursive step.</p

Inositol Phosphate Accumulation in Vivo Provides a Measure of Muscarinic M₁ Receptor Activation

The rationale for using M₁ selective muscarinic acetylcholine receptor activators for the treatment of cognitive impairment associated with psychiatric and neurodegenerative disease is well-established in the literature. Here, we investigate measurement of inositol phosphate accumulation, an end point immediately downstream of the M₁ muscarinic acetylcholine receptor signaling cascade, as an in vivo biochemical readout for M₁ muscarinic acetylcholine receptor activation. Five brain penetrant M₁-subtype selective activators from three structurally distinct chemical series were pharmacologically profiled for functional activity in vitro using recombinant cell calcium mobilization and inositol phosphate assays, and a native tissue hippocampal slice electrophysiology assay, to show that all five compounds presented a positive allosteric modulator agonist profile, within a narrow range of potencies. In vivo characterization using an amphetamine-stimulated locomotor activity behavioral assay and the inositol phosphate accumulation biochemical assay demonstrated that the latter has utility for assessing functional potency of M₁ activators. Efficacy measured by inositol phosphate accumulation in mouse striatum compared favorably to efficacy in reversing amphetamine-induced locomotor activity, suggesting that the inositol phosphate accumulation assay has utility for the evaluation of M₁ muscarinic acetylcholine receptor activators in vivo. The benefits of this in vivo biochemical approach include a wide response window, interrogation of specific brain circuit activation, an ability to model responses in the context of brain exposure, an ability to rank order compounds based on in vivo efficacy, and minimization of animal use

HeLa Cells infected with Timer-CVB3 slowly change fluorescence from green to red as determined by flow cytometric analysis.

<p>HeLa cells were either mock-infected or infected with eGFP-CVB (moi = 01), dsRed-CVB3 (moi = 0.1), or Timer-CVB3 (moi = 0.01 or 0.1). After the indicated times post-infection, the cells were isolated, centrifuged and resuspended in a solution of 4% paraformaldehyde in 1× PBS for fixation overnight. The cells were then centrifuged and resuspended in 0.1% BSA/1× PBS, and the cell solutions were stored at 4°C until analyzed by flow cytometry. Quadrants were set based in mock-infected control samples.</p