An enhanced median filter for removing noise from MR images

In this paper, a novel decision based median (DBM) filter for enhancing MR images has been proposed. The method is based on eliminating impulse noise from MR images. A median-based method to remove impulse noise from digital MR images has been developed. Each pixel is leveled from black to white like gray-level. The method is adjusted in order to decide whether the median operation can be applied on a pixel. The main deficiency in conventional median filter approaches is that all pixels are filtered with no concern about healthy pixels. In this research, to suppress this deficiency, noisy pixels are initially detected, and then the filtering operation is applied on them. The proposed decision method (DM) is simple and leads to fast filtering. The results are more accurate than other conventional filters. Moreover, DM adjusts itself based on the conditions of local detections. In other words, DM operation on detecting a pixel as a noise depends on the previous decision. As a considerable advantage, some unnecessary median operations are eliminated and the number of median operations reduces drastically by using DM. Decision method leads to more acceptable results in scenarios with high noise density. Furthermore, the proposed method reduces the probability of detecting noise-free pixels as noisy pixels and vice versa

Discovery of Enzymatic Targets of Transcriptional Activators via <i>in Vivo</i> Covalent Chemical Capture

The network of activator protein-protein interactions (PPIs) that underpin transcription initiation is poorly defined, particularly in the cellular context. The transient nature of these contacts and the often low abundance of the participants present significant experimental hurdles. Through the coupling of <i>in vivo</i> covalent chemical capture and shotgun LC-MS/MS (MuDPIT) analysis, we can trap the PPIs of transcriptional activators in a cellular setting and identify the binding partners in an unbiased fashion. Using this approach, we discover that the prototypical activators Gal4 and VP16 target the Snf1 (AMPK) kinase complex via direct interactions with both the core enzymatic subunit Snf1 and the exchangeable subunit Gal83. Further, we use a tandem reversible formaldehyde and irreversible covalent chemical capture approach (TRIC) to capture the Gal4-Snf1 interaction at the Gal1 promoter in live yeast. Together, these data support a critical role for activator PPIs in both the recruitment and positioning of important enzymatic complexes at a gene promoter and represent a technical advancement in the discovery of new cellular binding targets of transcriptional activators

Δ<i>sylC</i> bacteria loose motility during infection.

<p>GFP-expressing WT and Δ<i>sylC</i> bacteria were monitored by confocal microscopy before infiltration (A), after infiltration (B), in the exudate of infected leaves (C), and after co-infiltration with or without SylA (D). All motile bacteria are marked with a star. The percentages of motile bacteria over a 2 s timeframe are indicated on the bottom, with standard deviations for n = 5. See supplemental data for the movies and details. Bacteria are 1–2 µm long. These results are representative of three independent experiments. *, motile bacterium.</p

SylA diffuses and suppresses SA-mediated immunity in adjacent tissue.

<p>(<b>A</b>) RhSylA spread through the vasculature. A 1-µl aliquot of 2 mM RhSylA or 0.1% DMSO (mock) was applied at a wound site, and a fluorescence image was taken 2 h later. Scale bar, 1 mm. Arrowheads indicate wound inoculation sites. (<b>B</b>) RhSylA targets the proteasome in adjacent tissue. A 1-µl aliquot of 2 mM SylA was applied to an inoculation site and preincubated for 30 min. Subsequently, 1 µl of 2 mM RhSylA was added and incubated for another 2 h or 6 h. Proteins were extracted from tissue at 1–10 mm from the application site, and labeled proteins were detected by fluorescence scanning. *, background signal. (<b>C</b>) SylA targets the proteasome in adjacent tissue. A 1-µl aliquot of 1 mM SylA was applied at a wound site and incubated for 4 h. The application site was removed, extracts from adjacent tissues were labeled with MVB072, and fluorescently labeled proteins were detected. (<b>D</b>) Procedure for assaying wound entry by Δ<i>sylC</i>-GFP bacteria in adjacent tissue. Leaves of WT <i>N. benthamiana</i> were infiltrated with 50 µM SylA and 0.25% DMSO (E), 10⁵ WT bacteria or water (F), and the infiltrated region was marked. After 1 h (for SylA infiltration) or 1 d (for bacterial infiltration), Δ<i>sylC</i>-GFP bacteria were inoculated at a site 5 mm outside the infiltrated area. Wound entry was scored 5 d later by fluorescence microscopy. (<b>E</b>) SylA promotes wound entry by Δ<i>sylC</i>-GFP bacteria at a distance from the infiltrated region. (<b>F</b>) WT bacteria promotes wound entry at a distance from the infiltrated region. (<b>G</b>) Representative example of distant colonization of Δ<i>sylC</i>-GFP bacteria when inoculated next to areas infiltrated with WT-GFP bacteria. WT-GFP bacteria were infiltrated at 10⁵ bacterial cells/mL (lower right, bordered by dashed line). One day later, Δ<i>sylC</i>-GFP bacteria were inoculated at 5 mm from the infiltrated region. The picture was taken 5 d later. WT-GFP bacteria did not spread outside the infiltrated zone, but their presence promoted wound entry by Δ<i>sylC</i>-GFP in adjacent tissue. Arrowheads indicate colonies of Δ<i>sylC</i>-GFP in tissues adjacent to the wound inoculation site. (<b>H</b>) Procedure for assaying adjacent colonization by WT-GFP bacteria in adjacent tissue. Leaves of WT <i>N. benthamiana</i> were infiltrated with 50 µM SylA, 0.25% DMSO (H), 10⁵ WT bacteria or water (I), and the infiltrated region was marked. After 1 h (for SylA infiltration) or 1 d (for WT bacteria infiltration), WT-GFP bacteria were inoculated at 5 mm outside the infiltrated area and the plant was sprayed with 300 µM BTH or water. Wound entry was scored 5 d later by fluorescence microscopy. (<b>I</b>) SylA promotes wound entry in BTH-treated tissue at a distance from the infiltrated region. (<b>J</b>) SylA-producing WT bacteria promotes wound entry in BTH-treated tissue at a distance from the infiltrated region. (<b>E, F, I, J</b>) Error bars represent SEM of four independent biological replicates, each with 12 wound inoculations. P-values determined using the Student's <i>t</i>-test are indicated.</p

<i>NahG</i> blocks immunity in adjacent tissues and only partially promotes wound entry by <i>ΔsylC</i> bacteria.

<p>(<b>A</b>) Reduced wound entry by WT bacteria when inoculated next to Δ<i>sylC</i>-infiltrated regions is absent in <i>NahG</i> plants. Leaves of WT and <i>NahG</i>-transgenic <i>N. benthamiana</i> plants were infiltrated with WT or Δ<i>sylC</i> bacteria, and GFP-expressing WT PsyB728a bacteria were inoculated 1 d later at 0.5 cm from the border of the infiltrated region. Wound entry was monitored 5 d later by fluorescence microscopy. Error bars represent SEM of four independent experiments, each with 12 wound inoculations. P-values determined using the Student's t-test are indicated. (<b>B–C</b>) The Δ<i>sylC</i> mutant can colonize adjacent tissues in <i>NahG</i>-transgenic plants, though less than WT bacteria. WT and Δ<i>sylC</i> mutant bacteria were inoculated in WT and <i>NahG</i>-transgenic <i>N. benthamiana</i> plants, and wound entry was scored after 5 d by fluorescence microscopy. Error bars represent SEM of four independent experiments, each with 12 inoculations. P-values determined using the Student's t-test are indicated. (<b>C</b>) Representative pictures of colonization by WT or Δ<i>sylC</i> bacteria at 5 dpi in WT or <i>NahG</i>-transgenic plants. Fluorescence pictures were converted into inverted greyscale for better visibility. Scale bar, 1 mm.</p

SylA targets the proteasome of <i>N. benthamiana</i>.

<p>(<b>A</b>) MVB072 labels the catalytic proteasome subunits in <i>N. benthamiana</i> leaf extracts. Leaf extract of <i>N. benthamiana</i> was incubated with or without MVB072, an epoxomicin-based probe carrying both biotin and BODIPY. Biotinylated proteins were purified and detected by in-gel fluorescence scanning. (<b>B</b>) Proteins identified by mass spectrometry. In-gel trypsin digests (dashed areas) were analyzed by tandem mass spectrometry. Identified peptides are underlined in the sequences of the β1, β2, and β5 catalytic subunits of the proteasome. None of these peptides were found in the no-probe-control. No peptides were from the propeptide (grey) or the mature N-terminus, containing the catalytic Thr (bold). (<b>C</b>) SylA targets the proteasome of <i>N. benthamiana</i>. Leaf extracts of <i>N. benthamiana</i> were preincubated with 200 µM epoxomicin (Epox.) or SylA for 30 min and then labeled for 2 h with 2 µM MVB072 or RhSylA. Labeled proteins were detected by in-gel fluorescence scanning, and proteins were stained with Coomassie blue. (<b>D</b>) Structures of SylA and SylAsat. SylA has a Michael system in the ring that reacts with the catalytic Thr residues of the proteasome. This Michael system is absent in SylAsat due to saturation of the double bond. (<b>E</b>) SylAsat does not fully inhibit the proteasome. Leaf extracts were preincubated with 50 µM SylA and SylAsat and labeled with MVB072. Proteins were separated on protein gels and detected by fluorescence scanning and Coomassie blue staining. (<b>F</b>) Concentration dependency of proteasome inhibition by SylA. Leaf extracts were incubated with various SylA concentrations and then labeled with MVB072. Proteins were analyzed on a protein gel using fluorescence scanning and Coomassie blue staining.</p

Δ<i>sylC</i>-induced immune responses are weak compared to ETI/NHR responses.

<p>(<b>A</b>) Increased callose deposition by both WT and Δ<i>sylC</i> strains expressing the HopQ1-1 effector. Leaves were infiltrated with 2×10⁷ bacteria/mL, and fluorescent callose spots were quantified per 0.56 mm² after aniline blue staining at 24 hpi. Error bars indicate SEM of 20 technical replicates. These experiments were repeated three times with similar results. (<b>B</b>) Increased oxidative burst by both WT and Δ<i>sylC</i> strains expressing the HopQ1-1 effector. Leaves were infiltrated with 2×10⁸ bacterial cells/mL, and reactive oxygen species (ROS) were measured at 15 hpi in leaf discs floating on 200 µM MOPS containing L-012 for 20 min. The reduced ROS level of Δ<i>sylC</i>(EV)-infiltrated tissue compared to WT(EV)-infiltrated tissue was due to the late time point and the fact that ROS levels are transient. Error bars indicate SEM of eight technical replicates. These experiments were repeated three times with similar results. (<b>C</b>) Reduced bacterial growth of both WT and Δ<i>sylC</i> bacteria expressing the HopQ1-1 effector. Leaves were infiltrated with 2×10⁵ bacterial cells/mL in <i>N. benthamiana</i>, and bacterial growth was measured at 0, 4, and 7 dpi. Error bars represent standard deviation of four biological replicates. The experiment was repeated four times with similar results. (<b>D</b>) HopQ1-1-expressing WT and Δ<i>sylC</i> bacteria do not cause symptoms when infiltrated at low densities. Leaves were infiltrated at 2×10⁵ bacterial cells/mL, and pictures were taken at 7 dpi. (<b>E</b>) Cell death triggered by HopQ1-1-expressing WT and Δ<i>sylC</i> bacteria is blocked by the calcium transport inhibitor lanthanum chloride and the ATPase inhibitor sodium vanadate. Leaves were co-infiltrated with 1×10⁸ bacteria/mL with 50 µM lanthanum chloride or 1 µM sodium vanadate, and pictures were taken at 24 hpi.</p

SylA is necessary and sufficient for wound entry of PsyB728a and PsyB301D.

<p>(<b>A</b>) Exogenous SylA complements wound entry of the SylA-deficient Δ<i>sylC</i> strain of PsyB728a. <i>N. benthamiana</i> leaves were infiltrated with 50 µM SylA or 0.25% DMSO and wound-inoculated 1 h later. Colonization was scored at 5 dpi by fluorescence microscopy. (<b>B</b>) SylA biosynthesis is necessary for wound entry by PsyB301D. GFP-expressing PsyB301D and derived mutants in the SylA biosynthesis clusters (Δ<i>sylC</i>, Δ<i>sylD</i>, and Δ<i>sylA-E</i>) were wound-inoculated and scored at 5 dpi by fluorescence microscopy. (<b>C</b>) Exogenous SylA complements wound entry of SylA-deficient strains of PsyB301D. <i>N. benthamiana</i> leaves were preinfiltrated with 50 µM SylA or 0.25% DMSO and wound-inoculated 1 h later. Colonization was scored at 5 dpi by fluorescence microscopy. (<b>D</b>) The SylA biosynthesis cluster complements wound entry by PsyB301D. The Δ<i>sylA-E</i> mutant of PsyB301D was transformed with cosmid pPL3syl carrying the SylA biosynthesis cluster. GFP-expressing derivatives were wound-inoculated, and colonization was scored at 5 dpi by fluorescence microscopy. (<b>A–D</b>) GFP-expressing strains were wound-inoculated into <i>N. benthamiana</i> leaves, and colonization of tissue adjacent to the wound site was scored at 5 dpi by fluorescence microscopy. The photographs at the bottom show representative pictures taken by fluorescence microscopy at 5 dpi. Error bars indicate SEM of four independent experiments, each with 12 inoculations. P-values determined using the Student's <i>t</i>-test are indicated.</p

SylA blocks SA-mediated immunity.

<p>(<b>A</b>) SA signaling is blocked by SylA but not SylAsat. <i>N. benthamiana</i> leaves were infiltrated with 50 µM SylA or SylAsat and sprayed with 300 µM BTH. RNA was isolated from infiltrated tissues 6 h after BTH treatment and used as a template for semi-quantitative RT-PCR with gene-specific primers for <i>PR1a</i> and <i>Actin</i>. (<b>B</b>) Dose-dependent inhibition of BTH-induced <i>NbPR1a</i> expression by SylA. Leaves were infiltrated with various concentrations of SylA and sprayed 6 h later with 300 µM BTH. RNA was isolated from infiltrated tissues 6 h after BTH treatment and used as a template for semi-quantitative RT-PCR with gene-specific primers for <i>PR1a</i> (30 cycles) and <i>Actin</i> (24 cycles). (<b>C</b>) SylA blocks BTH-induced acquired resistance. <i>N. benthamiana</i> leaves were infiltrated with or without SylA and immediately sprayed with BTH. The Δ<i>sylC</i> mutant bacteria were infiltrated with 2×10⁵ bacteria/mL 6 h after SylA/BTH treatment, and bacterial populations were determined at 0 and 3 d after inoculation. Error bars represent SEM of four independent bacterial counts. This experiment was repeated three times with similar results. (<b>D</b>) SylA-producing bacteria grow better in BTH-treated tissues than Δ<i>sylC</i> bacteria. WT and Δ<i>sylC</i> bacteria were infiltrated into <i>N. benthamiana</i> leaves at 2×10⁵ bacteria/mL, and plants were sprayed with 300 µM BTH 6 h later. Bacterial populations were determined at 0, 3, and 7 dpi. Error bars represent SEM of four independent bacterial counts. This experiment was repeated two times with similar results. (<b>E</b>) SylA promotes wound entry in BTH-treated tissue. Leaves of <i>N. benthamiana</i> plants were infiltrated with 50 µM SylA or 0.025% DMSO. After 1 h, the infiltrated area was wound-inoculated with GFP-expressing WT or Δ<i>sylC</i> bacteria and sprayed with 300 µM BTH or water. Wound entry was scored at 5 dpi by fluorescence microscopy. Error bars represent SEM of four independent experiments. P-values determined using the Student's <i>t</i>-test are indicated.</p

SylA-deficiency triggers immune responses at primary infection sites.

<p>(<b>A</b>) Time course of cell death induced by WT and Δ<i>sylC</i> strains. Leaves were stained with trypan blue at various days after infiltration. Scale bar, 10 mm. (<b>B</b>) Cell death spreads from zones infected with WT but not Δ<i>sylC</i> bacteria. Leaves were infiltrated and stained with trypan blue at 7 dpi. (<b>C</b>) Cell death induced by Δ<i>sylC</i> is blocked by the calcium transport inhibitor lanthanum chloride and the ATPase inhibitor sodium vanadate. Leaves were co-infiltrated with 1×10⁸ bacteria/mL with 50 µM lanthanum chloride or 1 µM sodium vanadate, and pictures were taken at 24 hpi. (<b>D</b>) Early host cell death induced by SylA-deficient Δ<i>sylC</i> bacteria is preceded by callose deposition. Leaves were stained for callose at 24 hpi, examined by fluorescence microscopy, and depicted with equal settings. The callose spots per 0.56 mm² were quantified and printed below the picture with the SEM (n = 3). Scale bar, 0.1 mm. (<b>E</b>) Early host cell death induced by SylA-deficient Δ<i>sylC</i> bacteria is preceded by SA accumulation. <i>N. benthamiana</i> plants were infiltrated with 10⁵ bacteria/mL of WT and Δ<i>sylC</i> bacteria, and SA concentrations were measured at 3 and 24 hpi. Error bars represent SEM of three technical replicates. Student's t-test: P = 0.21 (3 hpi) and P = 0.096 (24 hpi). The experiment was repeated twice with similar results. (<b>F</b>) Early host cell death induced by SylA-deficient Δ<i>sylC</i> bacteria is preceded by upregulated transcript levels of the hypersensitive cell death marker <i>Hin1</i>. Semi-quantitative RT-PCR was performed on mRNA isolated at 24 hpi. (<b>A–F</b>) Bacteria were infiltrated with 2×10⁵ bacteria/mL into mature leaves of <i>N. benthamiana</i> and analyzed at 24 hpi unless stated otherwise.</p