On the Lab. II building site

425 special CDSs in PCN033 (XLSX 29 kb

Additional file 1 of TGFβ1-induced hedgehog signaling suppresses the immune response of brain microvascular endothelial cells elicited by meningitic Escherichia coli

Additional file 1

SphK2-S1P-S1P₂ mediates meningitic <i>E</i>. <i>coli</i> penetration of the BBB <i>in vitro</i> and <i>in vivo</i>.

<p>(A) S1P generation was significantly higher in HBMEC incubated with wild-type RS218 compared with the triple mutant deleted of <i>ompA</i>, <i>fimH</i> and <i>nlpI</i>. Correspondingly, the sphingosine level was lower in HBMEC incubated with wild-type RS218 than those incubated with the triple deletion mutant. * <i>p</i><0.05. (B) Structures of (<i>S</i>)-FTY720-vinylphosphonae (SphK1 and SphK2 inhibitor), (<i>R</i>)-FTY720-methyl ether (selective SphK2 inhibitor), RB-032 and RB-033 (selective SphK1 inhibitors), and RB-034 (inactive analogue). (C) Both (<i>S</i>)-FTY720-vinylphosphonae (SphK1 and SphK2 inhibitor, shown as (<i>S</i>)-FTY-Pn) and (<i>R</i>)-FTY720-methyl ether (SphK2 inhibitor, shown as ROME) significantly inhibited RS218 invasion of HBMEC, while the SphK1 inhibitors (RB-032 and RB-033) and inactive analogue (RB-034) did not exhibit any inhibition. ** <i>p</i><0.01. The inhibitors were all used at 10 μM. (D) (<i>R</i>)-FTY720-methyl ether inhibited <i>E</i>. <i>coli</i> RS218 invasion of HBMEC in a dose-dependent manner. ** <i>p</i><0.01. (E) <i>E</i>. <i>coli</i> RS218 activated SphK2 in a time-dependent manner in HBMEC, while such activation was abolished by pretreatment with 10 μM (<i>R</i>)-FTY720-methyl ether. ** <i>p</i><0.01. (F) <i>E</i>. <i>coli</i> penetration into the brain was significantly less in SphK2 ^−/− mice compared with wild-type mice. In contrast, the levels of bacteremia did not differ between the two groups of mice. (G) JTE-013 (S1P₂ antagonist) significantly inhibited <i>E</i>. <i>coli</i> invasion of HBMEC, while VPC23019 (S1P₁ and S1P₃ antagonist) did not exhibit any inhibition. ** <i>p</i><0.01. (H) The mutants with deletion of <i>ompA</i>, <i>fimH</i>, or <i>nlpI</i> as well as the triple mutant (<i>ΔompAΔfimHΔnlpI</i>) induced significantly lower levels of SphK2 activation in HBMEC, compared with wild-type RS218. ** <i>p</i><0.01.</p

SphK2-S1P-S1P₂ is upstream of EGFR activation in meningitic <i>E</i>. <i>coli</i> invasion of HBMEC and contributes to HB-EGF mediated transactivation of EGFR.

<p>(A) Activation of SphK2 in response to RS218 did not differ between HBMEC with and without gefitinib pretreatment. * <i>p</i><0.05, ** <i>p</i><0.01. (B) SphK2 activation was not affected in HBMEC expressing dominant-negative EGFR, while EGFR activation was, as expected, abolished in HBMEC expressing dominant-negative EGFR. Activation of c-Src occurred in response to <i>E</i>. <i>coli</i> in vector-transfected HBMEC, but did not occur in HBMEC expressing dominant-negative EGFR. ** <i>p</i><0.01. (C) JTE-013 (S1P₂ antagonist) inhibited EGFR activation in response to <i>E</i>. <i>coli</i> in HBMEC. ** <i>p</i><0.01. (D) Real-time PCR analysis of the expression of EGFR ligands in response to wild-type <i>E</i>. <i>coli</i> RS218 or the triple deletion mutant in HBMEC. Representative results from three independent assays are shown. GAPDH was used as an endogenous reference. (E) Pretreatment of HBMEC with JTE-013 or (<i>R</i>)-FTY720-methyl ether (shown as ROME) prevented HB-EGF up-regulation (analyzed by real-time PCR) in response to RS218. (F) Pretreatment of HBMEC with CRM197 prevented EGFR activation in response to RS218. ** <i>p</i><0.01. (G) CRM197 dose-dependently inhibited RS218 invasion of HBMEC, while only the highest dosage of CRM197 significantly affected HBMEC invasion by the triple mutant. ** <i>p</i><0.01. (H) The release of HB-EGF from HBMEC infected with the triple deletion mutant for up to 4 h was below the detection limit, while HB-EGF release was significantly increased by approximately 3-fold from the cells infected with wild-type RS218 at 4 h, ** <i>p</i><0.01.</p

S1P and EGFR promote meningitic <i>E</i>. <i>coli</i> invasion of HBMEC monolayer <i>via</i> exploiting c-Src.

<p>(A) Association of c-Src with EGFR in response to <i>E</i>. <i>coli</i> in HBMEC, as shown by co-immunoprecipitation of HBMEC lysates with an anti-EGFR antibody. (B) c-Src activation occurred in response to <i>E</i>. <i>coli</i> in a time-dependent manner in HBMEC, but was abolished by pretreatment with gefitinib. (C) Pretreatment of HBMEC with PP2 (Src inhibitor) exhibited a dose-dependent inhibition of <i>E</i>. <i>coli</i> RS218 invasion. ** <i>p</i><0.01. (D) <i>E</i>. <i>coli</i> RS218 invasion was significantly reduced in HBMEC expressing the dominant-negative Src construct, pEGFP-N1-Src-DN, compared with the vector (pEGFP-N1)-transfected HBMEC. ** <i>p</i><0.01. (E, F) Pretreatment of HBMEC with JTE-013 (S1P₂ antagonist) inhibited c-Src activation in response to <i>E</i>. <i>coli</i> (E), while pretreatment with PP2 (Src inhibitor) did not affect SphK2 activation (F). * <i>p</i><0.05, ** <i>p</i><0.01. (G) <i>E</i>. <i>coli</i> activation of EGFR and SphK2 was not affected in HBMEC expressing dominant-negative c-Src, while c-Src activation was, as expected, abolished in HBMEC transfected with dominant-negative c-Src compared with vector control-transfected HBMEC. ** <i>p</i><0.01.</p

Schematic representation of the S1P-EGFR signaling pathway in meningitic <i>E</i>. <i>coli</i> invasion of HBMEC.

<p>Meningitic <i>E</i>. <i>coli</i> penetration of the BBB follows the microbial-host interactions contributing to HBMEC invasion, via exploiting specific host cell signaling molecules. During the HBMEC invasion, meningitic <i>E</i>. <i>coli</i> strains activate SphK2, which catalyzes the synthesis of S1P from sphingosine. S1P is then secreted outside and acts on S1P receptor S1P₂. S1P interaction with S1P₂ is involved in the activation of EGFR, as well as the up-regulation and release of EGFR-related ligand HB-EGF, which is proteolytically processed by metalloproteinases. The released HB-EGF binds to the extracellular ligand-binding domain of EGFR and leads to tyrosine phosphorylation of the EGFR cytoplasmic kinase domain. This SphK2-S1P-S1P₂-EGFR cascade induces the activation of c-Src tyrosine kinase, an intracellular mediator that has been shown to regulate host cell actin cytoskeleton rearrangements, leading to <i>E</i>. <i>coli</i> invasion of HBMEC.</p

Meningitic <i>E</i>. <i>coli</i> exploits EGFR for its penetration of the BBB <i>in vitro</i> and <i>in vivo</i>.

<p>(A) The EGFR-selective inhibitor gefitinib inhibits meningitic <i>E</i>. <i>coli</i> RS218 invasion of HBMEC in a dose-dependent manner, but does not affect its adhesion. * <i>p</i><0.05, ** <i>p</i><0.01. (B) Bacterial growth was not affected by the treatment with gefitinib. Overnight bacterial culture was 1:100 transferred into fresh medium with or without gefitinib at indicated concentrations, and further incubated for 2 h. Viable bacterial counts were determined by series dilution and plating at 30 min interval. (C) Gefitinib did not lead to an inhibition of cell proliferation when used at the indicated concentrations. (D) Meningitic <i>E</i>. <i>coli</i> invasion of the EGFR knock-out HBMEC (KO#35) was significantly decreased compared to the invasion of the control cells. * <i>p</i><0.05. (E) A time-dependent activation of EGFR occurs in response to <i>E</i>. <i>coli</i> RS218 in HBMEC. The ratio of p-EGFR and EGFR was calculated based on densitometry analysis. ** indicates the difference was significant compared to time 0 (<i>p</i><0.01). (F) EGFR mRNA transcription levels did not change in response to <i>E</i>. <i>coli</i> RS218 in HBMEC, as assessed by real-time PCR analysis. GAPDH was used as the endogenous reference. Representative results from three individual experiments are shown. (G) EGFR protein expression levels were not affected in response to <i>E</i>. <i>coli</i> RS218 in HBMEC. Actin was probed in the same lysate and used as a loading control. (H) RS218 invasion was significantly reduced in HBMEC transfected with the dominant-negative EGFR construct pcDNA-EGFR-GGS compared with pcDNA3.1 control vector-transfected HBMEC. ** <i>p</i><0.01. (I) <i>E</i>. <i>coli</i> RS218 penetration into the brain was significantly inhibited by administration of gefitinib (10 mg/kg) compared with vehicle treatment. In contrast, the magnitudes of bacteremia did not differ between the recipients of gefitinib and vehicle control. * <i>p</i> <0.05. (J) Co-localization of <i>E</i>. <i>coli</i> strain RS218 and EGFR is demonstrated in HBMEC. Scale bar = 10 μm.</p

DataSheet_1_Genetic variation in morphological traits in cotton and their roles in increasing phosphorus-use-efficiency in response to low phosphorus availability.docx

Phosphorus (P) is an essential macronutrient required for fundamental processes in plants. Trait plasticity is crucial for plant adaptation to environmental change. Variations in traits underlie diverse phosphorus (P) acquisition strategies among plants. Nevertheless, how the intraspecific plasticity and integration of morphological traits contribute to Phosphorus-Use-Efficiency (PUE) in cotton is unknown. In this study, 25 morphological traits were evaluated in 384 cotton genotypes grown with low P (LP, 10μmol. L−1) and normal nutrition (CK, 500μmol. L−1) to assess the genetic variability of morphological traits and their relationship to phosphorus use efficiency. Results revealed a large genetic variation in mostly morphological traits under low P. Significant enhancement in root traits and phosphorus efficiency-related traits like PUE was observed at LP as compared to CK conditions. In response to low P availability, cotton genotypes showed large plasticity in shoot and total dry biomass, phosphorus, and nitrogen efficiency-related traits (i.e., phosphorus/nitrogen use efficiency, phosphorus/nitrogen uptake efficiency), and most root traits, but a limited response in root dry biomass, taproot length, root surface area, root volume, and SPAD value. In addition, significant correlations were observed between PUtE (phosphorus uptake efficiency), NUE (nitrogen use efficiency), TDB (total dry biomass), and RTD (root tissue density) with PUE under both P supply level and phosphorus stress index, which may be a key indicator for improving PUE under LP conditions. Most root traits are most affected by genotypes than nutrition level. Conserved PUE is more affected by the nutrition level than the genotype effect. Principal component analysis depicted the comprehensive indicators under two P supply conditions were mainly reflected in root-related traits and morphological indicators such as dry matter biomass. These results indicate that interspecific variations exist within these cotton genotypes and traits. Our study provides suggestions for future research to enhance the ability of the earth system model to predict how crops respond to environmental interference and provide target quality for cotton breeding in phosphorus-deficient areas.</p

DataSheet_2_Genetic variation in morphological traits in cotton and their roles in increasing phosphorus-use-efficiency in response to low phosphorus availability.xlsx

Phosphorus (P) is an essential macronutrient required for fundamental processes in plants. Trait plasticity is crucial for plant adaptation to environmental change. Variations in traits underlie diverse phosphorus (P) acquisition strategies among plants. Nevertheless, how the intraspecific plasticity and integration of morphological traits contribute to Phosphorus-Use-Efficiency (PUE) in cotton is unknown. In this study, 25 morphological traits were evaluated in 384 cotton genotypes grown with low P (LP, 10μmol. L−1) and normal nutrition (CK, 500μmol. L−1) to assess the genetic variability of morphological traits and their relationship to phosphorus use efficiency. Results revealed a large genetic variation in mostly morphological traits under low P. Significant enhancement in root traits and phosphorus efficiency-related traits like PUE was observed at LP as compared to CK conditions. In response to low P availability, cotton genotypes showed large plasticity in shoot and total dry biomass, phosphorus, and nitrogen efficiency-related traits (i.e., phosphorus/nitrogen use efficiency, phosphorus/nitrogen uptake efficiency), and most root traits, but a limited response in root dry biomass, taproot length, root surface area, root volume, and SPAD value. In addition, significant correlations were observed between PUtE (phosphorus uptake efficiency), NUE (nitrogen use efficiency), TDB (total dry biomass), and RTD (root tissue density) with PUE under both P supply level and phosphorus stress index, which may be a key indicator for improving PUE under LP conditions. Most root traits are most affected by genotypes than nutrition level. Conserved PUE is more affected by the nutrition level than the genotype effect. Principal component analysis depicted the comprehensive indicators under two P supply conditions were mainly reflected in root-related traits and morphological indicators such as dry matter biomass. These results indicate that interspecific variations exist within these cotton genotypes and traits. Our study provides suggestions for future research to enhance the ability of the earth system model to predict how crops respond to environmental interference and provide target quality for cotton breeding in phosphorus-deficient areas.</p

Additional file 2: Table S2. of Genome analysis and in vivo virulence of porcine extraintestinal pathogenic Escherichia coli strain PCN033

Core genome content used to construct phylogenetic tree (XLSX 225 kb