A Multi-Robot Cooperation Framework for Sewing Personalized Stent Grafts

This paper presents a multi-robot system for manufacturing personalized medical stent grafts. The proposed system adopts a modular design, which includes: a (personalized) mandrel module, a bimanual sewing module, and a vision module. The mandrel module incorporates the personalized geometry of patients, while the bimanual sewing module adopts a learning-by-demonstration approach to transfer human hand-sewing skills to the robots. The human demonstrations were firstly observed by the vision module and then encoded using a statistical model to generate the reference motion trajectories. During autonomous robot sewing, the vision module plays the role of coordinating multi-robot collaboration. Experiment results show that the robots can adapt to generalized stent designs. The proposed system can also be used for other manipulation tasks, especially for flexible production of customized products and where bimanual or multi-robot cooperation is required.Comment: 10 pages, 12 figures, accepted by IEEE Transactions on Industrial Informatics, Key words: modularity, medical device customization, multi-robot system, robot learning, visual servoing, robot sewin

Type VI secretion system contributes to Enterohemorrhagic <i>Escherichia coli</i> virulence by secreting catalase against host reactive oxygen species (ROS)

<div><p>Enterohemorrhagic <i>Escherichia coli</i> (EHEC) is one major type of contagious and foodborne pathogens. The type VI secretion system (T6SS) has been shown to be involved in the bacterial pathogenicity and bacteria-bacteria competition. Here, we show that EHEC could secrete a novel effector KatN, a Mn-containing catalase, in a T6SS-dependent manner. Expression of <i>katN</i> is promoted by RpoS and OxyR and repressed by H-NS, and <i>katN</i> contributes to bacterial growth under oxidative stress <i>in vitro</i>. KatN could be secreted into host cell cytosol after EHEC is phagocytized by macrophage, which leads to decreased level of intracellular reactive oxygen species (ROS) and facilitates the intramacrophage survival of EHEC. Finally, animal model results show that the deletion mutant of T6SS was attenuated in virulence compared with the wild type strain, while the deletion mutant of <i>katN</i> had comparable virulence to the wild type strain. Taken together, our findings suggest that EHEC could sense oxidative stress in phagosome and decrease the host cell ROS by secreting catalase KatN to facilitate its survival in the host cells.</p></div

The T6SS of EHEC does not show antibacterial activity <i>in vitro</i>.

<p>(A) The positive control of competition assays. <i>A</i>. <i>baylyi</i> ADP1 was used as a prey strain to be killed by the predator strains (<i>P</i>. <i>aeruginosa</i> PAO1, Δ<i>retS</i> or Δ<i>ppkA</i>). The predator strains were individually mixed with <i>A</i>. <i>baylyi</i> ADP1 in 20:1 ratio, and 5 μl of the mixture was spotted on LB agar plate. After incubated at 37°C for 2.5 h, bacterial spots were cut out and the cells were resuspended in 1 ml 1× PBS. The suspensions were diluted serially in 1× PBS, and 5 μl of the suspensions was spotted on the selective LB agar plates, followed by 16 h incubation at 30°C. PAO1 indicates <i>P</i>. <i>aeruginosa</i> PAO1; Δ<i>retS</i> indicates <i>P</i>. <i>aeruginosa</i> PAO1 Δ<i>retS</i>, a T6SS-activated (T6SS⁺) strain; Δ<i>ppkA</i> indicates <i>P</i>. <i>aeruginosa</i> PAO1 Δ<i>ppkA</i>, a T6SS-inactivated (T6SS^-) strain. EHEC T6SS⁺ (B) and T6SS^- (C) can not be killed by T6SS⁺ <i>P</i>. <i>aeruginosa</i>. (D) EHEC T6SS⁺ can not kill T6SS⁺ <i>P</i>. <i>aeruginosa</i>. Δ<i>hns</i> indicates EHEC Δ<i>hns</i>, a T6SS-activated strain. (E) T6SS⁺ EDL933 can not kill T6SS^- <i>E</i>. <i>coli</i> strain MG1655.</p

H-NS inhibits the expression of the T6SS genes of EHEC.

<p>(A) Transcription of T6SS genes in the wild type EHEC (WT) and <i>hns</i> deletion mutant (Δ<i>hns</i>). The WT and Δ<i>hns</i> were cultured to an OD₆₀₀ = 1.0 in LB broth at 37°C. Cultures were harvested, and total RNA was isolated. The relative expression levels of z0254, z0264, z0267 were analyzed by qPCR. 16S rRNA was used as the reference gene. Error bars represented SD from at least three independent experiments. ***, P<0.001, ANOVA analysis. (B) The transcription of T6SS genes in the <i>hns</i> deletion mutant and complementation strains. Δ<i>hns</i> harboring either plasmid pACYC184 or pACYC184-<i>hns</i> were cultured to an OD₆₀₀ = 1.0 in LB liquid at 37°C. Cultures were harvested followed by total RNA isolation. The relative expression levels of z0254, z0264(<i>hcp-2</i>) and z0267 were analyzed by qPCR. 16S rRNA was used as the reference gene. Error bars represented SD from at least three independent experiments. ***, P<0.001, ANOVA analysis. (C) The secretion of Hcp in EHEC. Δ<i>hns</i> and ΔT6SS harboring pQE80YX1-z0264 with a His-tag sequence fusion at the C-terminus were cultured to an OD₆₀₀ = 1.0 in LB broth at 37°C. The pellet and supernatant fractions of the cultures were analyzed by Western blot using anti-His tag antibody. Three biological repeats were performed. (D) Formation of discrete ClpV–GFP foci. The WT and ΔT6SS harboring pQE80-z0254<i>-gfp</i> were cultured to an OD₆₀₀ = 1.0 in LB liquid at 37°C. Cells were collected and resuspended in 1× PBS to an OD₆₀₀ = 10. The resuspended cells were mixed with <i>E</i>. <i>coli</i> strain MG1655 at the ratio of 10:1 and transferred to the agarose pad. After incubation in 37°C for 30 min, ClpV-GFP foci were observed by fluorescence microscopy. 100 cells were analyzed, and the final percentages were obtained from three independent experiments. (E) The intracellular survival of the WT and ΔT6SS in RAW264.7 macrophages. RAW264.7 cells were incubated with the WT or ΔT6SS at an MOI of 10 for 30 min and then chased in the presence of 100 μg/ml gentamicin for 2 h to kill extracellular bacteria. Cells were then incubated for 20 h in the presence of 25 μg/ml gentamicin. Lysates were then plated to count viable intracellular bacteria. Percent bacterial survival was calculated based on viable counts (CFU/ml) relative to that at 2.5 h post-infection. Error bars represented SD from at least three independent experiments. ***, P<0.001, Student’s t-test analysis.</p

Schematic diagram of the genetic organization of the type VI secretion system (T6SS) in EHEC O157:H7 strain EDL933.

<p>Annotation of T6SS cluster genes of the EHEC strain EDL933. The database of Clusters of Orthologous Groups of proteins (COGs) was obtained from the National Center of Biotechnology Information (<a href="http://www.ncbi.nlm.nih.gov/COG/new/" target="_blank">http://www.ncbi.nlm.nih.gov/COG/new/</a>).</p

T6SS contributes to EHEC virulence in mouse model.

<p>(A) ΔT6SS has lower virulence in mice model compared with the WT. The streptomycin pre-treated BALB/c mice were intragastrically infected with the WT or its derived mutants at the inoculation of 10¹⁰ CFU. The percentage of surviving animals (8 mice per group) on each day was calculated. ***, P<0.001, Log-rank analysis. (B) Body weight changes of BALB/c mice infected by the WT, ΔT6SS or <i>ΔkatN</i>. The average body weight change percentage of surviving animals (8 mice per group) on each day was calculated. Error bars represented SD from at least three independent experiments. ***, P<0.001; ns, Not significant, ANOVA analysis. (C) The colonization of the WT, ΔT6SS or <i>ΔkatN</i> in mice gut. At the indicated times, fecal samples were collected, homogenized, diluted and plated on SMAC agar plates to determine the numbers of the WT or its derived mutants. The log₁₀ means of CFU per gram of feces for each group of 8 mice are presented for each time point. Error bars represented SD from at least three independent experiments. ***, P<0.001; ns, Not significant, ANOVA analysis.</p

The secretion of KatN is dependent on T6SS in EHEC.

<p>(A) Verification of the T6SS effector candidates by Western blot. The WT and ΔT6SS bearing pQE80-z1921(<i>katN</i>) or z5583 or z0873 with a His-tag sequence fusion at the C-termini were cultured to an OD₆₀₀ = 1.0 in LB broth at 37°C. The pellet and supernatant fractions of the cultures were analyzed by Western blot using the anti-His tag and anti-RpoA monoclonal antibodies. RpoA was used as an internal control. Three biological repeats were performed. (B) z0254 is required for the secretion of KatN. The WT, Δz0254 and ΔT6SS harboring pQE80-z1921(<i>katN</i>) with a His-tag sequence fusion at the C-terminus were cultured to an OD₆₀₀ = 1.0 in LB broth at 37°C. The pellet and supernatant fractions of the cultures were analyzed by Western blot using the anti-His tag and anti-RpoA monoclonal antibodies. RpoA was used as an internal control. Three biological repeats were performed. (C) The β-lactamase fusion assay of KatN. The WT, Δz0254 and ΔT6SS bearing pCX340 or pCX-z1921(<i>katN</i>) were cultured to an OD₆₀₀ = 1.0 in LB broth at 37°C. The β-lactamase activities in the supernatants of the cultures were detected by monitoring optical density change at 486 nm. The β-lactamase activity data (means ± SD) represented the results from duplicate samples of three biological repeats. ***, P<0.001, ANOVA analysis. (D) TEM-1 β-lactamase translocation assay. RAW264.7 cells were infected with EHEC strain EDL933 (WT) or ΔT6SS bearing pCX340 or pCX-<i>katN</i>, and visualized by fluorescence microscopy with excitation at 409 nm or 488 nm after being treated by CCF2-AM. Emission due to CCF2-AM can be viewed as green fluorescence at 520 nm, whereas disruption of CCF2-AM by Bla fusion protein activity results in emission at 447 nm (blue fluorescence). One representative field was shown for each strain, and the assay was conducted at least twice. (E) The secretion of KatN is confirmed by Western blot using the anti-KatN antibody. The WT, Δz0254, Δz0254 bearing pACYC184-z0254 were cultured to an OD₆₀₀ = 1.0 in LB broth at 37°C. The pellet and supernatant fractions of the cultures were analyzed by Western blot using the anti-KatN and anti-RpoA antibodies. RpoA was used as an internal control. Three biological repeats were performed.</p

The role and transcriptional regulation of <i>katN</i>.

<p>(A) KatN contributes to the response of EHEC to oxidative stress. The WT, Δ<i>katN</i> and Δ<i>katN</i>-c were cultured in LB broth supplemented with H₂O₂ at final concentrations of 0, 1, 2, 4 and 6 mM at 37°C. The survival percentages were calculated by using the values OD₆₀₀ of cultures after 7 h incubation. The measurements were repeated three times and a representative experiment was shown. (B) The transcription of <i>katN</i> is promoted by RpoS and OxyR. Logarithmic-phase (OD₆₀₀ = 1.0) cultures (LB broth, 37°C) of the WT, Δ<i>rpoS</i>, Δ<i>oxyR</i> and ΔT6SS were divided into two aliquots followed by 1 mM H₂O₂ treatment or without H₂O₂ treatment. The cells were then harvested after 30 min and total RNA were isolated. The relative expression level of <i>katN</i> was analyzed by qPCR. 16S rRNA was used as the reference gene. Error bars represented SD from at least three independent experiments. **, P<0.01; ***, P<0.001; ns, Not significant, ANOVA analysis. (C) The expression of KatN is regulated by RpoS and OxyR. Logarithmic-phase (OD₆₀₀ = 1.0) and stationary-phase (OD₆₀₀ = 4.0) cultures (LB broth, 37°C) of the WT, Δ<i>rpoS</i> and Δ<i>oxyR</i> were divided into two aliquots followed by 1 mM H₂O₂ treatment or without H₂O₂ treatment. The cells were then harvested after 30 min and boiled with SDS sample buffer. The samples were separated in 12% SDS-PAGE followed by Western blot analysis using the anti-KatN and anti-RpoA monoclonal antibodies. RpoA was used as a loading control. Three biological repeats were performed. (D) KatN and other catalase genes expression in the WT and <i>hns</i> deletion mutant. Logarithmic-phase (OD₆₀₀ = 1.0) cultures (LB broth, 37°C) of the WT and Δ<i>hns</i> were harvested for total RNA isolation. The relative expression levels of <i>katN</i>, <i>katE</i> and <i>katG</i> were analyzed by qPCR. 16S rRNA was used as the reference gene. Error bars represented SD from at least three independent experiments. **, P<0.01; ***, P<0.001, ANOVA analysis. (E) H-NS inhibits the expression of KatN. Logarithmic-phase (OD₆₀₀ = 1.0) and stationary-phase (OD₆₀₀ = 4.0) cultures (LB broth, 37°C) of the WT and Δ<i>hns</i> were harvested and separated on 10% SDS-PAGE. KatN expression was analyzed by Western blot using the anti-KatN and anti-RpoA monoclonal antibodies. RpoA was used as a loading control. Three biological repeats were performed.</p

KatN is crucial for the survival of EHEC in macrophages.

<p>(A) The intracellular survival of the WT, Δ<i>katN</i> and Δ<i>katN-</i>c in RAW264.7 macrophages. RAW264.7 cells were incubated with the WT, Δ<i>katN</i> or Δ<i>katN</i> harboring pACYC184-<i>katN</i> (Δ<i>katN</i>-c) at an MOI of 10 for 30 min and then chased in the presence of 100 μg/ml gentamicin for 2 h to kill extracellular bacteria. Cells were then incubated for 20 h in the presence of 25 μg/ml gentamicin. Lysates were then plated to count viable intracellular bacteria. Percent bacterial survival was calculated based on viable counts (CFU/ml) relative to that at 2.5 h post-infection. All graphs displayed relative mean ± SD of at least three independent experiments. ***, P<0.001, ANOVA analysis. (B) The intracellular survival of the WT, Δ<i>katN</i> and Δ<i>katN-c</i> in primary peritoneal macrophages. Primary peritoneal macrophage cells isolated from BALB/c mice were incubated with the WT, Δ<i>katN</i> or Δ<i>katN</i>-c, then treated as described above. Error bars represented SD from at least three independent experiments. **, P<0.01; ns, Not significant, ANOVA analysis. (C) The expression of the T6SS genes and <i>katN</i> in macrophages. RAW264.7 cells were infected with the WT at an MOI of 50. After 8 h incubation, cells were lysed and total bacterial RNA was isolated. The relative expression levels of z0254, z0264, z0266, and z0267 were analyzed by qPCR. 16S rRNA was used as the reference gene. Error bars represented SD from at least three independent experiments. ***, P<0.001, ANOVA analysis. (D) KatN is secreted into the cytoplasm of macrophages <i>via</i> T6SS. RAW264.7 cells were infected with the WT or its derived mutants (ΔT6SS, Δ<i>katN</i>, Δ<i>katN</i>-c) at an MOI of 10. After 40 min incubation, cells were fixed with 4% para-formaldehyde and incubated with anti-O157 LPS and anti-KatN antibodies. Cell nuclei were counterstained with DAPI. Samples were analyzed by a laser-scanning confocal microscope. The red spots represented bacterial signals and the green spots represented KatN signals in the cytoplasm of RAW264.7 cells. Three biological repeats were performed. Scale bar equals 10 μm. (E) KatN contributes to the decrease of ROS level in macrophage cells. Hyper3-transfected RAW264.7 cells were infected with the WT or its derived mutants (Δ<i>katN</i> and Δ<i>katN-</i>c) at an MOI of 10. After 40 min incubation, cells were quantified at excitation wavelengths of 405 nm and 488 nm by a laser-scanning confocal microscope. The ratios of 488 nm/405 nm were the averages of at least 10 cells. At least three biological repeats were performed. SD was derived from at least three independent experiments. **, P<0.01, ANOVA analysis. Scale bar equals 10 μm.</p