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

A Screen of <i>Coxiella burnetii</i> Mutants Reveals Important Roles for Dot/Icm Effectors and Host Autophagy in Vacuole Biogenesis

<div><p><i>Coxiella burnetii</i> is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create a pathogen-occupied vacuole remain largely unknown. Here, we conducted a visual screen on an arrayed library of <i>C. burnetii</i> NMII transposon insertion mutants to identify genes required for biogenesis of a mature <i>Coxiella</i>-containing vacuole (CCV). Mutants defective in Dot/Icm secretion system function or the PmrAB regulatory system were incapable of intracellular replication. Several mutants with intracellular growth defects were found to have insertions in genes encoding effector proteins translocated into host cells by the Dot/Icm system. These included mutants deficient in the effector proteins Cig57, CoxCC8 and Cbu1754. Mutants that had transposon insertions in genes important in central metabolism or encoding tRNA modification enzymes were identified based on the appearance filamentous bacteria intracellularly. Lastly, mutants that displayed a multi-vacuolar phenotype were identified. All of these mutants had a transposon insertion in the gene encoding the effector protein Cig2. Whereas vacuoles containing wild type <i>C. burnetii</i> displayed robust accumulation of the autophagosome protein LC3, the vacuoles formed by the <i>cig2</i> mutant did not contain detectible amounts of LC3. Furthermore, interfering with host autophagy during infection by wild type <i>C. burnetii</i> resulted in a multi-vacuolar phenotype similar to that displayed by the <i>cig2</i> mutant. Thus, a functional Cig2 protein is important for interactions between the CCV and host autophagosomes and this drives a process that enhances the fusogenic properties of this pathogen-occupied organelle.</p></div

The effector Cig2 is important for CCV fusion with autophagosomes.

<p>(A) HeLa cells were infected with <i>C. burnetii</i> NMII, <i>cig2</i>::Tn or <i>cig2</i>::Tn pFLAG:Cig2 for 5 days and stained with anti-LC3 (green) and anti-<i>Coxiella</i> (red) antibodies. (B) The association of LC3 with the CCV was quantified by observing 100 infected cells per sample in 3 independent experiments. (C) LC3 and actin immunoblots from HeLa cells infected with parental <i>C. burnetii</i> NMII, the <i>cig2</i>::Tn mutant, the parental <i>C. burnetii</i> NMII producing either RavZ or RavZ_C258A, or uninfected cells. Cells were untreated (top image), or treated (bottom image) with bafilomycin A1 and rapamycin for 2 h prior to lysis. (D) J774A.1 cells were infected with <i>C. burnetii</i> NMII, <i>cig2</i>::Tn, or <i>cig2</i>::Tn pFLAG:Cig2 for 36 h before loading with 50 µg/ml DQ Green BSA for 16 h. Micrographs show phase contrast images to reveal vacuoles and the green fluorescence that results from DQ Green BSA degradation by proteases in lysosomal compartments.</p

The Dot/Icm effector Cig57 is required for efficient intracellular replication of <i>C. burnetii</i>.

<p>(A) Intracellular replication of <i>C. burnetii</i> NMII (black squares), <i>cig57</i>::Tn (black triangles) and <i>cig57</i>::Tn pFLAG-Cig57 (open circles). The fold-increase in GEs relative to the inoculum was determined by <i>dotA</i>-specific qPCR and is represented here as the mean ± SD of 3 independent infections at days 1, 3, 5 and 7 post-infection. (B,C) Representative micrographs of HeLa cells infected for either 3 days (B) or 5 days (C) with <i>C. burnetii</i> NMII, <i>cig57</i>::Tn or <i>cig57</i>::Tn pFLAG-Cig57. Cell with stained with anti-<i>Coxiella</i> antibody (red), anti-LAMP1 antibody (green) and Hoechst dye (blue). Note that the vacuoles containing <i>cig57</i>::Tn were smaller and contained few bacteria at each time point compared to the control NMII strain or the complemented <i>cig57</i>::Tn pFLAG-Cig57 strain. Scale bars represent 10 µm.</p

The Dot/Icm system and the PmrAB system are essential for intracellular replication and delivery of <i>C. burnetii</i> effector proteins.

<p>(A) Indicated are the locations of transposon insertions in the chromosomal regions encoding the Dot/Icm system from 1559100 bp to 1591800 bp, and (B) the PmrAB two-component regulatory system from 1176500 to 1179500 bp. The site of each transposon insertion is represented by an arrow. The mutants that could not replicate intracellularly were assigned red arrows, mutants that produced normal CCVs were assigned black arrows, and mutants that formed small vacuoles were assigned grey arrows. (C) The plasmids pBlaM and pBlaM-77 were introduced into <i>C burnetii</i> NMII (black bars) and <i>pmrA</i>::Tn (grey bars) to determine whether the PmrAB system was essential for effector translocation. Cleavage of the fluorescent β-lactamase substrate CC4F-AM was determined by calculating the ratio of fluorescence at 460 nm to 535 nm relative to uninfected cells. The graph shows the mean ± SD calculated for three independent samples.</p

Transposon mutants of <i>C. burnetii</i> display different intracellular phenotypes.

<p>Transposon mutants were subject to a vacuole formation assay in which 96-well plates of HeLa 229 cells were infected, at an MOI of approximately 500, with individual transposon mutants. Following a 96 h infection period, the infection was fixed and stained with anti-<i>Coxiella</i> (red), anti-LAMP1 (green) and Hoechst dye (blue) and observed with low magnification fluorescence microscopy. (A) The parental strain <i>C. burnetii</i> NMII displayed a large CCV in the majority of HeLa cells. (B) A cohort of mutants showed no intracellular replication as demonstrated by <i>dotA</i>::Tn, (C) another category produced smaller replicative vacuoles such as that seen with <i>cig57</i>::Tn, (D) transposon insertions that disrupted <i>cig2</i> resulted in the appearance of multiple vacuoles in a single cell, and (E) a small proportion of mutants, such as <i>gidA</i>::Tn, displayed CCVs with an abnormal shape due to filamentous replication of the <i>C. burnetii</i>.</p

Autophagy is required for homotypic fusion of CCVs.

<p>(A) HeLa cells in which the indicated genes were silenced using siRNA were infected with <i>C. burnetii</i> and fixed 3 days post-infection before immunostaining with anti-<i>C. burnetii</i> (red) and anti-LAMP1 (green) antibodies. DNA was stained with Hoechst (blue). Shown are representative micrographs for mock-transfected cells and cells transfected with siRNA specific to human ATG5, ATG12, STX17, and STX18. (B) Single-cell quantification of <i>C. burnetii</i> vacuole counts in ATG5, ATG12, and STX17 siRNA-transfected HeLa cells compared to STX18-transfected cells and mock-transfected cells at 3 days post-infection. The percentages of infected cells with two or more <i>C. burnetii</i> vacuoles were counted. Shown is the mean ± SD from 3 independent experiments. (C) HeLa cells infected with <i>C. burnetii</i> expressing the <i>L. pneumophila</i> effector RavZ from a plasmid (3×FL-RavZ) were assayed 3 days after infection and scored for the multi-vacuolar phenotype. As controls, <i>C. burnetii</i> expressing empty vector (3×FL) or a point mutant of RavZ defective in autophagy inhibition (3×FL-RavZ_C258A) were used. Shown are representative fluorescent images for each <i>C. burnetii</i> infection. (D) Immunoblots from uninfected or <i>C. burnetii</i>-infected HeLa cell lysates showing bands for non-lipidated (LC3-I) and lipidated (LC3-II) forms of LC3 and actin. Cells were untreated (top images) or treated (bottom images) with bafilomycin A1 and rapamycin for 2 h prior to lysis. Cells were infected with a Dot/Icm-deficient strain of <i>C. burnetii</i> (<i>icmL</i>::Tn), infected with parental <i>C. burnetii</i> NMII (no vector), or <i>C. burnetii</i> NMII with vector alone or vectors producing the indicated RavZ proteins. (E) Single-cell quantification of <i>C. burnetii</i> vacuole counts in cells at 3 days post-infection. The percentages of infected cells with two or more <i>C. burnetii</i> vacuoles were counted. Shown is the mean ± SD from three independent experiments.</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

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

<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