Draft Genome Sequence of Gordonia lacunae BS2T

We report here the draft genome sequence of the soil bacterium Gordonia lacunae BS2T ( DSM 45085T JCM 14873T NRRL B-24551T), isolated from an estuary in Plettenberg Bay, South Africa. Analysis of the draft genome revealed that more than 40% of the secondary metabolite biosynthetic genes encode new compounds

A Novel Single-Labeled Fluorescent Oligonucleotide Probe for Silver(I) Ion Detection in Water, Drugs, and Food

Due to the high toxicity of silver­(I) ions, a method for the rapid, sensitive, and selective detection for silver­(I) ions in water, pharmaceutical products, and food is of great importance. Herein, a novel single-labeled fluorescent oligonucleotide (OND) probe based on cytosine–Ag­(I)–cytosine coordination and the inherent fluorescence quenching ability of the G-quadruplex is designed to detect silver­(I) ions. The formation of a hairpin structure in the OND–Ag­(I) complex brings the hexachloro fluorescein (HEX) labeled at the 5′-end of the OND probe close to the G-quadruplex located at the 3′-end of the OND probe, leading to a fluorescence quenching due to photoinduced electron transfer between HEX and the G-quadruplex. Through this method, silver­(I) ions can be detected quantitatively, the linear response range is from 1 to 100 nmol/L with a detection limit of 50 pmol/L, and no obvious interference occurs with other metal ions with a 10-fold concentration. This assay is simple, sensitive, and selective, and it can be used to detect silver­(I) ions in actual water, drug, and food samples

The effects of the net advancement speed of the cell edge on the contact time between the lamellipodium and a filopodia adhesion.

<p><b>A.</b> Scatter plot of the part of the contact time of a filopodia adhesion with the advancing lamellipodium, with respect to the net advancement speed of the cell edge. All data were for REF52-β3-integrin-EGFP cells spreading on FN coated glass. <i>Black:</i> maturing filopodia adhesions. <i>Red:</i> disassembling filopodia adhesions. Due to the fast net advancement speed of the cell edge, the disassembling filopodia adhesions demonstrated a reduced contact time with the advancing lamellipodia, which subsequently had very brief of no pausing at these filopodia adhesions. Open symbols correspond to the filopodia adhesions in B–b. <b>B. a.</b> A fast spreading REF52 fibroblast expressing β3-integrin-EGFP (green) on FN coated glass (5 min–9 min 30 s after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s013" target="_blank">Video S8</a>). The cell membrane was stained with the fluorophore DiI (red). <b>b.</b> Magnified views of the dashed rectangle regions (I and II) in a. The selected frames from the time-lapse sequences showed the disassembly (I, horizontal arrows) or steady maturation (II, vertical arrows) of the filopodia adhesions as a result of ongoing advancement or prolonged pausing of lamellipodium at the two filopodia adhesions respectively. <b>c.</b> The advancement of cell two edges are represented by the kymographs that were generated along the corresponding kymograph lines (colored arrows in a) from the membrane fluorescence signal at the two filopodia adhesions. The dashed lines indicate the advancing (yellow) and pausing (pink) phases of lamellipodia at the respective filopodia adhesions. The net advancement speeds of the cell edges were measured at the corresponding sections of the kymographs as indicated by the dashed yellow lines (B-c I, 161 nm/s; B–c II, 147 nm/s). Scale bars: 2 µm (B–b), 5 µm (B–a).</p

The actin cytoskeleton organization at filopodia adhesions as seen in ventral cell membrane samples.

<p>Cells were gently blasted open. While this might cause some local disruptions, the main purpose was to distinguish between molecular components that are weak versus strongly bound to the ventral site of the plasma membrane. The colors of stains denote the ventral cell membrane (red) and actin structures (green). Filopodia adhesions were identified (see method) by vinculin immunostaining (cyan, A–b, B–b), interference reflection signals (grey images, B), and β3-integrin-EGFP imaging (grey, C). <b>A and B.</b> Cell edge regions of the exposed ventral cell membranes from HFF cells (20 min (A) or 33 min (B) after plating on FN coated glass). The white square regions (A–a, B–a) were magnified respectively (b, c in A; b–f in B). In merged images, vinculin was alternatively false colored in red (A–c, B–e, B–f) for a better presentation of the colocalization of signals. Pink arrow or arrowheads denote the former filopodia actin bundles and their associated substrate adhesions. The cyan arrowheads indicate circumferential stress fibers with their surface anchorage (A) or tight surface association (B). The white arrow (A–c) indicates the separation between the filopodia adhesion in the cell lamellum and the distal section of the filopodium, which might be fracture that could have occurred during the sample preparation. <b>C.</b> The cell periphery of an exposed ventral cell membrane from a REF52-β3-integrin-EGFP cell (30 min after plating on FN coated glass) showed a β3-integrin cluster at the filopodium (pink arrows), circumferential stress fibers (cyan arrowheads) anchored to the substrate via the β3-integrin clusters formed at the side of this filopodia adhesion, and the sidewise widening of the filopodia β3-integrin cluster (pink arrowheads) following the connected thick circumferential stress fibers in the cell lamellum. Scale bar: 5 µm (A–b & c, B, C), 10 µm (A–a).</p

Growth kinetics of filopodia adhesions after MLCK inhibition (A, B) or on soft polyacrylamide gels (C, D).

<p>The β3-integrin-EGFP (green) expressing REF52 fibroblasts on FN coated glass (A, B) or polyacrylamide gel (C, D) surfaces were time-lapse tracked with confocal microscopy. The cell membrane was stained by the fluorophore DiI (red). <b>A and B.</b> Inhibition of MLCK by ML-7 suppressed the cyclic protrusions and retractions of lamellipodium and the growth of filopodia adhesions. <b>A.</b> Time-lapse montage (b) of the filopodium containing cell edge region (white rectangle, a) showed the loss of the cyclic protrusions and retractions of the lamellipodium in the proximity of the filopodium in ML-7 treated cells (25 min–30 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s009" target="_blank">Video S4</a>). <b>B.</b> Time-lapse montage (b) of the filopodia adhesion cell edge region (white rectangle, a) demonstrated the suppressed size growth of the filopodia β3-integrin adhesion in ML-7 treated cells. c. Comparison of the area (average of the tracked temporal states (0–600 s)) of the filopodia β3-integrin adhesions (7 adhesions, n = 17) in the ML-7 (30 or 40 µM) treated cells with that of the maturing filopodia β3-integrin adhesions (6 adhesions, n = 164) in the cells plated in normal culture media. Error bars correspond to the standard errors. <b>C and D.</b> The growth of filopodia adhesions in REF52-β3-integrin-EGFP cells on soft PAA substrates was associated with the restored cyclic protrusions and retractions of lamellipodia. The PAA substrate (7.4 kPa) was covalently coated with FN. <b>C.</b> Cell without stable surface adhesions (a, 103 min–113 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s010" target="_blank">Video S5</a>) exhibited significant ruffling as shown by the time-lapse montage of the cell edge (b). <b>D.</b> The filopodia adhesion (white rectangle, b) at the edge of a cell (white square in a, 121 min–136 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s011" target="_blank">Video S6</a>) matured in association with the restored cyclic protrusions and retractions of the lamellipodium (c). To better visualize lamellipodium activities, all montages were stretched vertically 2×. Scale bars: 5 µm (A, D–b), 10 µm (B, C, D–a). The width of a single image frame in the montage is indicated by the horizontal grey bar: A–b, 2.5 µm; B–b, 4.5 µm; C–b, 6.2 µm; D–c, 3 µm.</p

The suppressed growth of filopodia adhesions in cases of insufficient contact time with the lamellipodium.

<p><b>A.</b> Time-lapse montage of the cell edge region containing a disassembling filopodia adhesion in a β3-integrin-EGFP (green) expressing REF52 fibroblast on FN coated glass (40 min–53 min after plating, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s012" target="_blank">Video S7</a>). This dissembling filopodia adhesion was located in the same cell region and captured in the same image sequence as for the maturing filopodia adhesion analyzed in Fig. 1. The cell membrane was stained by the fluorophore DiI (red). The colored bars indicate the relation between the filopodia adhesion and the lamellipodium as in Fig. 2A. <i>Black segment:</i> filopodia adhesion before being reached by the advancing lamellipodium. <i>Yellow segment</i>: passing of the advancing lamellipodium. <i>Pink segment</i>: pausing of the lamellipodium at the distal end of the filopodia adhesion without net advancement. <i>Grey segment:</i> the disassembly of the filopodia adhesion inside the cell lamellum after its full separation from the advancing lamellipodium. <b>B.</b> Time traces of the length (a), width (b), area (c) and average fluorescence intensity (d) of the disassembling filopodia β3-integrin clusters (same color code as for the maturing filopodia adhesions in Fig. 2A–D). Growth trace 1 corresponds to the dissembling filopodia adhesion in A. <b>C.</b> Comparison of the total contact time with lamellipodia (the averaged sum of the advancing and pausing durations of lamellipodia at filopodia adhesions) between the disassembling filopodia adhesions (n = 6) and those matured into stable adhesions (n = 18). Error bars correspond to the standard errors.</p

Growth kinetics of β3-integrin-EGFP clusters of filopodia tip or base adhesions (A–D.).

<p>Each curve represented one filopodia adhesion as REF52-β3-integrin-EGFP cells spread on FN coated glass (cells after plating, 44–56 min (Tip a), 12–24 min (Tip b), 17–29 min (Tip c), 25–37 min (Tip c), 5–17 min (Base a), 7–19 min (Base b)). The length (A), width (B), area (C) and average fluorescence intensity (D, average = sum of grey value/number of pixels) were plotted for β3-integrin-EGFP clusters for filopodia tip adhesions (thin curves, representing 4 maturing filopodia adhesions in 3 cells) and filopodia base adhesions (thick opaque curves, representing 2 maturing filopodia adhesions in 1 cell). Curves Tip-a and Base-a corresponded to the filopodia adhesions shown in Fig. 1 and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s001" target="_blank">Fig. S1</a> respectively. The traces were color coded (similar to the colored zones in Fig. 1B) to indicate the different local movements (advancing or pausing) of the lamellipodium in relation to the growth of the filopodia adhesion (<i>black</i>: before being reached by lamellipodium; yellow: being passed by an advancing lamellipodium; <i>pink</i>: with the lamellipodium persisted at the distal end of the filopodia adhesion without net advancement). <b>E.</b> The spatial distribution of β3-integrins in filopodia adhesions. For the filopodia adhesions analyzed in A–D, the fluorescence intensity profiles were generated along the long axes of filopodia β3-integrin-EGFP clusters in the last frame of their tracked sequences, as indicated by the dashed arrow in the <i>Insert</i> (same as analyzed in Fig. 1; green: β3-integrin-EGFP; red: membrane). The range of the x axis for the intensity profile curves corresponded to the full lengths of filopodia adhesions between their distal and proximal ends. <b>F.</b> Vinculin recruitment to filopodia adhesions. The white squared cell edge region of a HFF cell (a, 20 min plated on FN coated glass) was magnified (b–f) in respective signals and their overlays. <b>G.</b> Scatter plot of average fluorescence intensities of vinculin clusters within filopodia adhesions with respect to their areas. The grey and pink data points corresponded to the filopodia adhesions before reached by lamellipodia or in contact with lamellipodia respectively. Open symbols correspond to the filopodia adhesions indicated by the arrowheads (in respective colors) in e and f. Scale bars: 2 µm (F–b, E insert), 10 µm (F–a).</p

Cycles of periodic protrusions and retractions of a lamellipodium in the proximity of maturing filopodia adhesions.

<p><b>A.</b> The β3-integrin-EGFP (green) expressing REF52 fibroblast on a FN coated glass surface was time-lapse tracked with confocal microscopy. The cell membrane was stained by the fluorophore DiI (red). <b>B.</b> Magnified views of the white rectangle region in A showed the filopodia adhesion at the start (left) and end (right) of the time-lapse tracking sequence (20 min–47 min after seeding, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107097#pone.0107097.s008" target="_blank">Video S3</a>). <b>C.</b> Time-lapse montages of the white rectangle region in A. At the time point indicated by the grey asterisk, we had to increase the laser (488 nm) intensity to compensate for photobleaching. To better visualize lamellipodium activities, the presented montage was stretched vertically 2×. <b>D.</b> Histograms of the distances, durations and speeds of the cyclic protrusions (black bars) and retractions (brown bars) of lamellipodia in proximity to filopodia adhesions. Values in the parenthesis gave the number of measurements (at 14 filopodia adhesions in 5 cells). Scale bars: 1 µm (B), 10 µm (A). The horizontal grey bar indicates the width of a single image frame in the montage in C (2.5 µm).</p

miRNAs with validated functions in mosquitoes.

<p>miRNAs with validated functions in mosquitoes.</p

Stratification of miRNA studies by the experimental approach based on mosquito species.

<p>Stratification of miRNA studies by the experimental approach based on mosquito species.</p