Additional file 1: of Comparison of nasopharyngeal and guttural pouch specimens to determine the optimal sampling site to detect Streptococcus equi subsp equi carriers by DNA amplification

Limit of detection for eqbE LAMP assay was determined via a “standard curve” as 0.005 ng of DNA using S. equi ATCC 33398. (DOCX 23 kb

Calculated values of conductance (G) for a K⁺ ion pulled through the KcsA channel using a step-wise pulling protocol.

<p>Values of <i>λ</i> are the center of the harmonic pulling potential, and values between <i>λ</i> = 7 Å and <i>λ</i> = 17 Å occur when the center of the pulling potential is within the selectivity filter of KcsA. A negative value of G simply implies negative work (see Materials and Method) corresponding to the attraction (not resistance) from the selectivity filter at a pulling step. But for all pulling steps, the total conductance is always positive because the total work and diffusion coefficients are always positive.</p

Histogram showing the frequency (probability) of finding the Na⁺ and K⁺ ions at each position (<i>z</i>) in the selectivity filter during step-wise pulling.

<p>The histogram is constructed from 25 pulling-step simulations for each ion. During relaxation of each pulling step, the positions of the pulled ions were collected every 100 fs and were put into bins of 0.01 Å width along the z-axis. Sites S0–S4 correspond to sites of stable K⁺ binding in the selectivity filter of KcsA. Stable Na⁺ positions are located in the plane of the carbonyl oxygen atoms that separate stable K⁺ binding sites. Note that Na⁺ ions are not stable within the selectivity filter at positions beyond the S2–S3 junction until they emerge from the channel at S0. Z = 0 corresponds to the center of mass of the protein-lipid-water system, within the central water-filled cavity in the transmembrane domain of the channel.</p

The number of water molecules within 3 Å of Na⁺ and K⁺ ions as they are pulled incrementally through the selectivity filter of KcsA from the cytoplasmic side of the membrane (<i>left</i>) to the extracellular surface of the membrane (<i>right</i>).

<p>On the x-axis, z = 0 corresponds to the center of mass of the system in the vestibule of the channel. The first data point on the left is a position in the vestibule below site S4. Site S4 is located at z≈7 Å. Values shown are means ± SD.</p

Z-Coordinates (Å) for Stable Positions for Na⁺ and K⁺ Ions After Each Pulling Step (Δλ = 1 Å).

<p>Na⁺ and K⁺ ions were pulled in increments (Δ<i>λ</i>) of 1 Å using a soft harmonic potential of 0.6 kcal/mol/Ų that has its minimum value at <i>λ</i>. At the start of the pulling sequence <i>λ</i> is centered at the center of mass of the simulation system, z = 0. As <i>λ</i> is incremented by Δ<i>λ</i>, the spatial coordinates of the Na⁺ and K⁺ ions are unconstrained during the relaxation period τ. The z-coordinate of the ions at the end of <i>τ</i> is shown in the Table. Multiple values in the Table reflect the different z-coordinates observed in the simulations. A histogram of the frequency of occurrence of the different values for z for each ion at all values of <i>λ</i> is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086079#pone-0086079-g003" target="_blank">Figure 3</a>.</p

Schematic diagram for a step-wise pulling protocol.

<p>(A) Control parameter <i>λ</i>, and (B) expectation value of an applied potential versus time, U(z, <i>λ</i>) = k(z–<i>λ</i>)²/2. Coordinate z is the position of an ion along the z-direction. The pulling in the direction of increasing <i>λ</i> is called forward; the opposite direction of pulling is called backward. (C) Comparison between profiles for PMF (<i>blue</i>) and free energy changes (</p><p><i>red</i></p>) computed using our method.<p></p

Positions of carbonyl oxygen atoms for amino acids T75–G79 in the KcsA selectivity filter during step-wise pulling of either Na⁺ (<i>top row</i>) or K⁺ (<i>bottom row</i>) from the vestibule toward the extracellular surface of the membrane.

<p>The y-axis is the frequency (probability) of finding the oxygen atoms at the position shown on the x-axis. Zero in the middle of the x-axis corresponds to the position of the atoms observed in the crystal structure. The carbonyl atoms corresponding to each amino acid in the tetrameric selectivity filter are shown in a single color, and each amino acid is represented by a different color (T75, <i>black</i>; V76, <i>green</i>; G77, <i>blue</i>; Y78, <i>red</i>; G79, <i>light blue</i>).</p

Experimental timelines.

<p>(A) Chronically catheterized mice (femoral artery and vein) were given three days to recover from surgery and infused intravenously (iv) with either saline or glucose for 24 hours prior to intra-arterial administration of either lipopolysaccharide (LPS; 1 mg/kg) or vehicle. Five hours after LPS or vehicle administration a frequently sampled intravenous glucose tolerance test (FSIVGTT) was performed over a two hour period. An iv glucose bolus of 1 g/kg D50 was given over approximately 15 sec (▲). Subsequently, multiple samples of either blood glucose and plasma insulin (Δ) or glucose alone (↑) were taken from the arterial catheter at the times identified. Red blood cells were spun down and re-infused into the mouse throughout the protocol to avoid anemia. (B) Mice underwent cecal ligation and puncture (CLP) immediately after chronic catheterization of the femoral artery and vein. Animals were infused intravenously with either saline or glucose upon completion of surgery and followed for 24 hrs. Blood glucose and plasma insulin (Δ) or blood glucose alone (↑) were sampled from the arterial catheter at the times identified.</p

Frequently sampled intravenous glucose tolerance test data.

<p>Glucose disposal curves in response to an iv injection of 1 g/kg D50 administered over 15 sec at time t = 0 are depicted for (A) mice receiving iv saline infusion and vehicle (Veh Sal, open diamonds) or iv saline infusion and intra-arterial lipopolysaccharide (1 mg/kg, LPS Sal, closed diamonds) and (B) iv glucose infusion and vehicle (Veh Glu, open triangles) or mice receiving iv glucose infusion and LPS (1 mg/kg, LPS Glu, closed triangles). The corresponding insulin response curves are shown for (C) Veh Sal and LPS Sal and (D) Veh Glu and LPS Glu groups.</p

Minimal model data from frequently sampled intravenous glucose tolerance test.

<p>Data are shown for (A) glucose tolerance (area under the glucose curve; AUCg), (B) insulin sensitivity (Si), (C) acute insulin response to glucose (AIRg), and (D) Disposition Index (DI = Si *AIRg) for four groups of animals receiving either iv saline infusion and vehicle (Veh Sal, open diamonds), iv saline infusion and intra-arterial lipopolysaccharide (1 mg/kg, LPS Sal, closed diamonds), iv glucose infusion and vehicle (Veh Glu, open triangles), or iv glucose infusion and LPS (1 mg/kg, LPS Glu, closed triangles). Group medians are indicated by gray lines. Statistical differences were determined by Kruskal-Wallis test with differences between individual means determined by post-hoc Wilcoxon rank-sum tests corrected for multiple comparisons using the Bonferroni method.</p