Evaluation and optimization of IgY spin column technology in the depletion of abundant proteins from human serum.

Serum depletion strategies are commonly implemented in order to remove abundant proteins, increasing the number of proteins detected in a biomarker study. The IgY spin columns used in this study bind 12 and 14 primate proteins, respectively. 1-D SDS-PAGE and 2-DE revealed a suboptimal performance of the IgY spin columns. However, modification of the manufacturer's protocol, subjecting samples to two rounds of depletion, improved the number of proteins resolved by 2-DE. With alteration of the manufacturer protocol, the Seppro(®) IgY14 spin column can produce depleted serum with an increased number of spots resolved by 2-DE compared to untreated serum.</p

Results of the fishery simulations of the assignment units identified using the top ranked 288 SNPs.

<p>A) 100% simulations, B) Mixed Stock Fishery simulation with equal proportions in each assignment unit, C) Mixed Stock Fishery simulation with proportions in each assignment unit based on productivity of that unit. In A and B, horizontal grey lines represent actual simulated proportions and points represent mean simulation proportion estimates. In C, black bars represent simulated proportions and light bars mean simulation proportion estimates. In all plots, bars represent 95% confidence intervals calculated over 1000 replicate simulations.</p

Assignment accuracy to river of hold-out set fish to training set reference sites using the top ranked 288 SNPs.

<p>Assignment accuracy to river of hold-out set fish to training set reference sites using the top ranked 288 SNPs.</p

Rivers (numbers) and associated sample collection sites (black circles).

<p>Rivers (numbers) and associated sample collection sites (black circles).</p

Multidimensional plot of pairwise D_A based on all SNPs.

<p>Red points are sites identified as outliers; green points are sites south of the Tweed on the East coast; orange points are Kyle of Sutherland sites (i.e. around sites 12 and 13 on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164327#pone.0164327.g001" target="_blank">Fig 1</a>) and blue points represent the remaining Scottish samples.</p

Steps taken in defining assignment units and accuracy of assignment to the units identified.

<p>Steps taken in defining assignment units and accuracy of assignment to the units identified.</p

Assignment units as defined by <i>k</i>-means clustering.

<p>A) Results of <i>k</i>-means clustering (insert) showing <i>k</i> = 7 as best cluster number, shown by differently coloured symbols. B) Final assignment units after combining rivers with reciprocal misassignments.</p

DCGs are released from SCs during mating, activating BMP signalling.

<p><b>A, B</b>. SCs from 6-day-old esgF/O^ts virgin males (A) and from males immediately after mating (B) were fixed and stained with an anti-pMad antibody (red) and DAPI (blue), revealing that the proportion of SCs with detectable nuclear pMad is higher in mated animals. <b>C, D.</b> Immediately after mating, living SCs (D) have less GFP-GPI-labelled DCGs than virgins (C). Image shows a single z-plane of gland stained with Lysotracker Red; not all compartments are in the focal plane. Note that the largest MVBL in (C; arrowhead) contains GFP, probably because of fusion between a DCG compartment and the MVBL [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006366#pgen.1006366.ref017" target="_blank">17</a>]. <b>E</b>. Graph shows proportion of SCs with nuclear pMad in 6-day-old virgin, and mated males (dissected 8 min into mating [Mid] and immediately after mating), and mated males expressing <i>dpp</i>-RNAi and Dad in SCs from eclosion onwards using the <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4</i> driver. <b>F</b>. Graph shows number of GFP-GPI-positive DCG compartments in 6-day-old virgin and mated males (using the <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI</i> driver line; Double is twice mated in 2 h), and at different times after single mating in control SCs. Compartments were also counted in SCs expressing <i>Snap24</i> RNAi post-eclosion in virgins and immediately after mating. Labelled compartments were counted using a complete z-series for each cell. Genotypes for images are: <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP/+; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4/+</i> (A, B); <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI/CyO</i> (C, D).***P<0.001, Kruskal-Wallis test, n>15. Scale bar for A-B is 20 μm and C-D is 10 μm.</p

Autocrine Dpp regulates SC growth, SV number, endolysosomal trafficking and exosome secretion.

<p><b>A</b>. Expression of <i>dpp</i>-RNAi during the first six days of adulthood using the esgF/O^ts driver reduces the size of SCs and their nuclei (green arrows) relative to MCs (red arrows). <b>B</b>. Relative SC:MC nuclear size for SCs expressing RNAis targeting <i>dpp</i> and <i>gbb</i>, or GFP-tagged Dpp and Gbb, revealing specific effects of Dpp on growth. <b>C</b>. <i>dpp</i>^<i>blk</i>-GAL4 drives expression of a UAS-coupled nuclear GFP exclusively in SCs of the AG. <b>D, E.</b> Mosaic expression of <i>dpp</i>-RNAi or Dpp-GFP in a subset of SCs has a stronger effect on nuclear growth in expressing cells (on–green arrows) than in non-expressing (off–red arrows; white dashed circle) SCs, although <i>dpp</i> knockdown also reduces growth in the latter. <b>F-K.</b> Co-expression of <i>dpp</i>-RNAi with CD63-GFP using the <i>dsx</i>-GAL4 driver (G) reduces non-acidic SV number (eg., marked by arrowhead) and increases GFP fluorescence in largest MVBL (arrow; stained with Lysotracker Red) compared to controls (F); the statistical analysis of these changes for two independent RNAis is shown in H and I respectively. Knockdown of <i>dpp</i> either results in a small increase in the size of the largest MVBL or no significant size change (J), and reduces exosome secretion (K). Confocal images are from fixed glands (A, C, D) stained with DAPI (blue) and for Fas3 (yellow) or from living glands (F, G). Genotypes for images are: <i>w; esg-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-FLP; UAS-GFP</i>_<i>nls</i> <i>actin>FRT>CD2>FRT>GAL4/P[TRiP</i>.<i>HMS00011]attP2</i> (A and mosaic in D; the esgF/O^ts driver was also used to generate data in E); <i>w; P[w</i>^<i>+</i> <i>UAS-GFP</i>_<i>nls</i><i>]; P[w</i>^<i>+</i> <i>dpp</i>^<i>blk</i><i>-GAL4]</i> (C); <i>w; UAS-CD63-GFP tub-GAL80</i>^<i>ts</i><i>; dsx-GAL4</i> combined with no other transgene (F) or <i>P[TRiP</i>.<i>HMS00011]attP2</i> (III) (G). ***P<0.001, Kruskal-Wallis test, n = 10. Scale bar for A, D is 20 μm, F, G, 10 μm, and for C, 50 μm.</p

Rapid replenishment of DCGs after mating is BMP-dependent.

<p><b>A</b>. Schematic representation of pulse-chase experiments shown in B-F, indicating the duration of GFP-GPI and <i>Dad</i> overexpression and timings of mating events. <b>B-D</b>. 6-day-old flies were shifted to 28.5°C for 24 h to allow expression of GFP-GPI in virgins (B) or in males mated 8 h after the start of the pulse (C). The number of GFP-GPI-labelled DCGs in SCs was reduced in virgin males co-expressing Dad (D). <b>E.</b> Graph shows a significant increase in the number of labelled DCGs if males are mated at 8h during a 24 h GFP-GPI pulse. The number of DCGs labelled in virgin and mated males is reduced if <i>Dad</i> is co-expressed. <b>F</b>. The increase in labelled compartments after mating is also reduced by Dad co-expression. The <i>w; spi-GAL4 tub-GAL80</i>^<i>ts</i> <i>UAS-GFP-GPI</i> line was used to generate data in E and F. *P<0.05, ***P<0.001, Kruskal-Wallis test, n = 15. Scale bar in B-D, 10 μm.</p