Journal: Nature chemical biology

Article Title: Monitoring methionine sulfoxide with stereospecific mechanism-based fluorescent sensors

doi: 10.1038/nchembio.1787

Figure Lengend Snippet: E. coli cells expressing MetSOx ( a ) or MetROx ( b ) were incubated with MetO for ~30 min, and then rinsed to remove the oxidant. Arrows indicate the addition (↓) of MetO and rinsing (↑) the cells for two representative experiments (Assay 1 and Assay 2). MetSOx ( c ) and MetROx ( d ) oxidized fractions calculated from (n = 3), using and , respectively, described in the “ online methods ” section.

Article Snippet: SoluBL21 TM E. coli (Gelantis, San Diego, CA) cells were transformed with an indicated expression vector and grown in Luria-Bertani containing ampicillin (50 μg.ml −1 ) at 37°C.

Techniques: Expressing, Incubation

Journal: Cellular immunology

Article Title: LPS Enhances CTB-INSULIN Induction of IDO1 and IL-10 Synthesis in Human Dendritic Cells

doi: 10.1016/j.cellimm.2019.03.003

Figure Lengend Snippet: Endotoxin Levels in Purified CTB-INS Protein Preparations

Article Snippet: E. coli BL-21 cells producing recombinant CTB-INS were lysed in 20 ml of Buffer Z (8.0 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0) by sonication on ice with a Sonic 60 Dismembrator (Fisher Sci.

Techniques: Purification

Journal: Cellular immunology

Article Title: LPS Enhances CTB-INSULIN Induction of IDO1 and IL-10 Synthesis in Human Dendritic Cells

doi: 10.1016/j.cellimm.2019.03.003

Figure Lengend Snippet: Panel (A), Human moDCs suspended in 2.0 mL of RPMI culture medium were treated with: 1) PBS as a negative control (NC), 2) LPS (1 μg/mL), 3) partially purified CTB-INS (10 μg/mL) isolated by nickel affinity column from E. coli BL-21 cells, containing residual levels of LPS, and 4) completely purified CTB-INS (10 μg/mL) treated with both Triton X-114 and Endotrap resin (T/E) to remove residual LPS. The levels of LPS were determined by Limulus amebocyte assay of all DC samples. Panel B, the DCs were cultured at 37°C for 24h and the amount of IDO1 protein synthesized in the DCs was identified by western blotting. Panel C, The activity of IDO1 synthesized in response to the different treatments was determined in all DC samples by kynurenine assay of the cell culture medium. The experimental data presented was from an individual donor representative of a total of three DC donors. The data for each measurement is expressed as the standard error of the mean of triplicate DC samples obtained from each donor. *p < 0.05, in comparison with the NC group.

Article Snippet: E. coli BL-21 cells producing recombinant CTB-INS were lysed in 20 ml of Buffer Z (8.0 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0) by sonication on ice with a Sonic 60 Dismembrator (Fisher Sci.

Techniques: Negative Control, Purification, Isolation, Affinity Column, Cell Culture, Synthesized, Western Blot, Activity Assay

Journal: Cellular immunology

Article Title: LPS Enhances CTB-INSULIN Induction of IDO1 and IL-10 Synthesis in Human Dendritic Cells

doi: 10.1016/j.cellimm.2019.03.003

Figure Lengend Snippet: Panel (A), are histograms generated by flow cytometric identification of DC costimulatory factors CD80, CD86 and maturation factor CD83 induced by CTB-INS fusion protein in the presence of residual LPS or absence of LPS endotoxin. The DCs were stained for surface expression of CD80, CD86 and CD83 and the costimulatory factor markers were determined by flow cytometric measurement (x-axis = MFI; y-axis = cell #). DC activation is expressed as mean fluorescence intensity (MFI). Panel (B), is a bar graph representing the data in panel A. Immature moDC activation is expressed as mean fluorescence intensity (MFI) after 24 h incubation of the DCs in RPMI culture medium containing 1μg/mL of LPS (red bar); 10 μg/mL of CTB-INS with residual LPS (green bar), 10 μg/mL of pure CTB-INS treated with both triton X-114 and Endotrap resin to remove residual LPS (blue bar) or PBS as a no treatment negative control (NC), (sky blue bar). The amount of LPS in each sample was determined. The experimental treatments are represented by the same colors used in panel (A). The data presented was taken from a representative donor from a total of three donors and is expressed as mean standard error for triplicate samples from the donor. *p < 0.05, in comparison with the NC group.

Article Snippet: E. coli BL-21 cells producing recombinant CTB-INS were lysed in 20 ml of Buffer Z (8.0 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0) by sonication on ice with a Sonic 60 Dismembrator (Fisher Sci.

Techniques: Generated, Staining, Expressing, Activation Assay, Fluorescence, Incubation, Negative Control

Journal: Cellular immunology

Article Title: LPS Enhances CTB-INSULIN Induction of IDO1 and IL-10 Synthesis in Human Dendritic Cells

doi: 10.1016/j.cellimm.2019.03.003

Figure Lengend Snippet: Panel A, Graphic representation of ELISA assay of DC secretion of the pro-inflammatory cytokine IL-12p70 detected in RPMI culture medium after 24 h treatment of moDCs with PBS (NC), 1μg/mL of LPS, 10 μg/mL of CTB-INS containing residual LPS, 10 μg/mL of CTB-INS treated with Triton X-114 and Endotrap resin to remove residual LPS. Panel B is a graphic representation of DC secretion of the anti-inflammatory cytokine IL-10. The error bars represent the standard deviation from the mean of three independent ELISA experiments. *p < 0.05, in comparison with the NC.

Article Snippet: E. coli BL-21 cells producing recombinant CTB-INS were lysed in 20 ml of Buffer Z (8.0 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0) by sonication on ice with a Sonic 60 Dismembrator (Fisher Sci.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Cellular immunology

Article Title: LPS Enhances CTB-INSULIN Induction of IDO1 and IL-10 Synthesis in Human Dendritic Cells

doi: 10.1016/j.cellimm.2019.03.003

Figure Lengend Snippet: Panel (A) is a graphic representation of quantitative real time-PCR (RT-qPCR) analysis of human IDO1 mRNA levels synthesized in untreated (NT) or LPS free CTB-INS (LPS = 0.005EU/mL) treated moDCs transfected with human IDO1-specific small interfering RNA (siIDO1), or a negative control siRNA (siC). IDO1 gene expression was normalized in comparison with β-actin gene expression. Panel (B) Western blot analysis of IDO1 protein synthesis in moDCs treated with CTB-INS and transfected with IDO1-specific siRNA (siIDO) or negative control (siC) using human anti-IDO1 as the primary antibody. Panel (C) Viability of CTB-INS treated human moDCs after transfection with IDO1-specific siRNA (siIDO). DC viability was measured as the percentage of CTB-INS treated DCs negative for uptake of viability dye FVD450. Panel (D) Graphic representation of siRNA inhibition of IDO1 effects on LPS free CTB-INS activation of DC costimulatory factor biosynthesis measured by flow cytometry (MFI). Panels (E and F) graphic representation of ELISA quantification of secreted IL-12p70 and IL-10 cytokines from LPS free CTB-INS treated moDCs. The graph bars represent the mean of three independent experiments. NT− = siIDO1 RNA transfected DCs in the absence of LPS free CTB-INS fusion protein treatment. NT+ = siIDO1 RNA transfected DCs treated with CTB-INS (LPS = 0.005 EU/mL). The DC samples were assayed in triplicate and the data represent the mean ± SD of three independent experiments. *p < 0.05, compared with the NT and **p < 0.05 in comparison with siC.

Article Snippet: E. coli BL-21 cells producing recombinant CTB-INS were lysed in 20 ml of Buffer Z (8.0 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0) by sonication on ice with a Sonic 60 Dismembrator (Fisher Sci.

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Synthesized, Transfection, Small Interfering RNA, Negative Control, Expressing, Western Blot, Inhibition, Activation Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Cellular immunology

Article Title: LPS Enhances CTB-INSULIN Induction of IDO1 and IL-10 Synthesis in Human Dendritic Cells

doi: 10.1016/j.cellimm.2019.03.003

Figure Lengend Snippet: Panel (A) Immunoblot detection of IDO1 levels in human moDCs treated with increasing amounts of LPS free CTB-INS. PBS treated DCs are a negative control (NC) for addition of CTB-INS. The amount of LPS in each CTB-INS treated DC sample was determined by the Limulus amebocyte lysate assay. Actin protein bands provide a loading control for DC protein in each well. Panel (B) graphic representation of costimulatory factors CD80, CD86 and CD83 synthesized on LPS free CTB-INS treated DCs determined by flow cytometric detection of fluorescent Ab binding to DC co-stimulatory factors (MFI). Panels (C and D) graphic representation of ELISA determination of pro-inflammatory (IL-12p70) and anti-inflammatory (IL-10) cytokines secreted by DCs into the culture medium following treatment of the DCs with increasing amounts of LPS free CTB-INS. Individual bars represent the mean and standard deviation (SD) of three independent ELISA cytokine determinations. *p < 0.05, in comparison with NC.

Article Snippet: E. coli BL-21 cells producing recombinant CTB-INS were lysed in 20 ml of Buffer Z (8.0 M urea, 100 mM NaCl, 20 mM HEPES, pH 8.0) by sonication on ice with a Sonic 60 Dismembrator (Fisher Sci.

Techniques: Western Blot, Negative Control, Synthesized, Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Molecular Cell

Article Title: Cryo-EM Structures of MDA5-dsRNA Filaments at Different Stages of ATP Hydrolysis

doi: 10.1016/j.molcel.2018.10.012

Figure Lengend Snippet:

Article Snippet: Escherichia coli BL21(DE3) cells , Merck , 69450.

Techniques: Recombinant, Activity Assay, Reporter Assay, Software, Single Particle, In Vitro

Journal: eLife

Article Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

doi: 10.7554/eLife.50928

Figure Lengend Snippet: ( A ) The scaffold used in structure determination of E. coli Crl-TAC. ( B, C ) The top and front view orientations of the cryo-EM density map ( B ) and structure model ( C ) of E. coli Crl–TAC. The RNAP, Crl and nucleic acids are presented as cartoon and colored as indicated in the color key. The density map is shown in gray envelop. ( D ) The cryo-EM density map (blue transparent surface) for Crl. ( E ) The cryo-EM density map (red transparent surface) for the upstream junction of the transcription bubble of promoter DNA and σ S . NT, non-template-strand promoter DNA; T, template-strand promoter DNA.

Article Snippet: The Ec Crl was overexpressed in E. coli BL21(DE3) cells (Novo protein, Inc) carrying pET28a-TEV-Crl.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

doi: 10.7554/eLife.50928

Figure Lengend Snippet: ( A ) Elution peaks of E. coli Crl–TAC from a size-exclusion column. ( B ) SDS-PAGE and ( C ) native-PAGE of the E. coli Crl–AC. The gel was first stained with SYBR Gold for nucleic acids and then with Coomassie Brilliant Blue for proteins.

Article Snippet: The Ec Crl was overexpressed in E. coli BL21(DE3) cells (Novo protein, Inc) carrying pET28a-TEV-Crl.

Techniques: SDS Page, Clear Native PAGE, Staining

Journal: eLife

Article Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

doi: 10.7554/eLife.50928

Figure Lengend Snippet: ( A ) The flowchart of data processing for the cryo-EM structure of E. coli Crl–TAC. ( B ) Representative cryo-EM micrograph. ( C ) Representative 2D classifications of E. coli Crl–TAC single particles. ( D ) Angular distribution of E. coli Crl–TAC particle projections. ( E ) The gold-standard Fourier shell correlation (FSC) of E. coli Crl–TAC. The dotted line represents the 0.143 FSC cutoff, which indicates a nominal resolution of 3.80 Å.

Article Snippet: The Ec Crl was overexpressed in E. coli BL21(DE3) cells (Novo protein, Inc) carrying pET28a-TEV-Crl.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

doi: 10.7554/eLife.50928

Figure Lengend Snippet: ( A ) Crl binds to the σ S 2 and the RNAP-β’ subunit. Crl is represented as a blue transparent surface and cartoon. ( B ) σ S is embedded into a shallow hydrophobic groove of Crl. The electrostatic potential surface of Crl was generated using APBS tools in Pymol. ( C ) The detailed salt-bridge interaction between the ‘R’ loop of Crl and the ‘specificity loop’ of σ S . Salt-bridge bonds are shown as blue dashed lines. ( D ) The N-terminal tail of Crl makes potential weak interactions with RNAP-β’ subunit, probably through R11 and S10. The colors are as in . ( E ) The yeast two-hybrid assay reveals the key interface residues (red) of Crl (left) or σ S (right). Mutating the key residues disrupts interactions between Crl and σ S . AD, the activation domain of GAL4; BD, the DNA-binding domain of GAL4. ( F ) The in vitro transcription assay shows that mutating most of the key interface residues of Crl (left) substantially impairs its transcription activation activity, and that mutating most of the key interface residues of σ S resulted in reduced response to Crl. ΔR-loop, replacing residue 43–51 with a ‘GSGS’ linker. ( G ) Protein sequence alignment of Crl from 72 non-redundant bacterial species. Filled circles indicate residues that are involved with interactions with σ S ; filled red circles indicate key contact residues. The residues are numbers as in E. coli Crl. NT, non-template-strand promoter DNA; T, template-strand promoter DNA.

Article Snippet: The Ec Crl was overexpressed in E. coli BL21(DE3) cells (Novo protein, Inc) carrying pET28a-TEV-Crl.

Techniques: Generated, Y2H Assay, Activation Assay, Binding Assay, In Vitro, Transcription Assay, Activity Assay, Residue, Sequencing

Journal: eLife

Article Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

doi: 10.7554/eLife.50928

Figure Lengend Snippet: ( A ) Structure superimposition of Crl proteins from crystal structure of Crl (PDB: 4Q11; cyan) and Crl in E. coli Crl-TAC (blue). ( B ) The σ S 2 inserts into a hydrophobic groove on surface of Crl. ( C ) The interaction between the ‘R’ loop of Crl and the ‘specificity’ loop of σ S 2 . The cryo-EM density maps for the side chains of the interface residues of Crl and σ S 2 are shown as in blue and green mesh, respectively. The C, N, and O atoms of Crl residues are shown in white, blue, and red, respectively. The C, N, and O atoms of σ S residues are shown in yellow, blue, and red, respectively. ( D ) Structure superimposition of our E. coli Crl–σ S –RPo complex and E. coli σ S –RPo complex (PDB 5IPM) reveals similar conformation of the specificity loop. ( E ) E. coli Crl interacts with σ S 2 in our structure. ( F ) Mycobacterium smegmatis RbpA interacts with σ A 2 in PDB 5TW1. ( G ) Caulobacter cresentus GcrA interacts with σ 70 2 in PDB 5YIX. The conserved region and the non-conserved region (NCR) of σ S 2 in ( E ), ( F ), and ( G ) are shown in yellow and orange, respectively.

Article Snippet: The Ec Crl was overexpressed in E. coli BL21(DE3) cells (Novo protein, Inc) carrying pET28a-TEV-Crl.

Techniques: Cryo-EM Sample Prep

Journal: eLife

Article Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

doi: 10.7554/eLife.50928

Figure Lengend Snippet: ( A ) Sequence alignment of six σ 70 -type E. coli σ factors. ( B ) The sequence alignment of the σ 70 subunit of ten representative bacterial species ( Proteus mirabilis , Escherichia coli , Salmonella enterica serovar Typhi, Yersinia enterocolitica , Shigella flexneri , Vibrio cholerae , Dickeya dadantii , Providencia stuartii , Xenorhabdus bovienii , and Photobacterium profundum ). ( C ) The sequence alignment of the σ S subunit of the same ten bacterial species. The residues are numbered as in E. coli .

Article Snippet: The Ec Crl was overexpressed in E. coli BL21(DE3) cells (Novo protein, Inc) carrying pET28a-TEV-Crl.

Techniques: Sequencing

Journal: eLife

Article Title: Crl activates transcription by stabilizing active conformation of the master stress transcription initiation factor

doi: 10.7554/eLife.50928

Figure Lengend Snippet:

Article Snippet: The Ec Crl was overexpressed in E. coli BL21(DE3) cells (Novo protein, Inc) carrying pET28a-TEV-Crl.

Techniques: Recombinant, Plasmid Preparation, Expressing, Cloning, Electron Microscopy