e coli j5 lps  (ATCC)

Journal: International Journal of Molecular Sciences

Article Title: Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification †

doi: 10.3390/ijms25073927

Figure Lengend Snippet: SDS-PAGE analysis of the different LPSs used in the present study. 1— Y. pestis ; 2— Y. enterocolitica ; 3— B. pertussis 1414; 4— B. pertussis A100; 5— E. coli 0119; 6— E. coli J5; 7— N. meningitidis ; 8— P. aeruginosa P3; 9— S. Typhimurium Ra; 10— R. pickettii ; 11—LPS-MW Standard. Staining was performed according to Tsai and Frash . Rough-type LPSs migrate in the low-masses level at about 4 kDa.

Article Snippet: E. coli J5 LPS was extracted from strain ATCC 43745.

Techniques: SDS Page, Staining

Journal: International Journal of Molecular Sciences

Article Title: Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification †

doi: 10.3390/ijms25073927

Figure Lengend Snippet: MALDI mass spectra obtained for tetra-acyl E. coli J5 lipid A ( A ) and hexa-acyl E. coli J5 lipid A ( B ). Corresponding lipid A structures ( C ). Comparison of LAL and HEK-blue hTLR-4 activities ( D ). Fatty acids other than 14:0-3-OH are presented with different colors.

Article Snippet: E. coli J5 LPS was extracted from strain ATCC 43745.

Techniques: Comparison

Journal: Biology

Article Title: Lysophosphatidylcholine Acetyltransferase 2 ( LPCAT2 ) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype

doi: 10.3390/biology13050314

Figure Lengend Snippet: Efficiency of LPCAT2 gene silencing in RAW264.7 macrophages with or without LPS infection. ( A ) The melt curve for the LPCAT2 amplicon was obtained by reverse transcribing LPCAT2 mRNA to cDNA and amplifying cDNA with real-time qPCR. Using R/ggplot2 (method = “gam”, formula = y~s (x, bs = “cs”)), the smooth curve was created from pooled data from all LPCAT2 RT-qPCR experiments (≥3 independent experiments). ( B ) A control group of RAW264.7 macrophages were transiently transfected with negative siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. After incubation, the cells were lysed for RNA extraction and RT-qPCR. ( C ) A group of RAW264.7 macrophages was transiently transfected with LPCAT2 siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. ( D ) LPCAT2 gene expression in the control group is compared with that in the silenced LPCAT2 group. Bars and error bars represent the mean ± standard error of the mean. **— p < 0.01, ***— p < 0.001, ****— p < 0.0001.

Article Snippet: Semi-rough (Rc) LPS— E. coli J5 and Rough (Re) LPS— E. coli K12 D32m4—was purchased from List Biological Laboratories, Eastbourne, UK.

Techniques: Infection, Amplification, Quantitative RT-PCR, Control, Transfection, Incubation, RNA Extraction, Gene Expression

Journal: Biology

Article Title: Lysophosphatidylcholine Acetyltransferase 2 ( LPCAT2 ) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype

doi: 10.3390/biology13050314

Figure Lengend Snippet: Transcriptional effects of silencing LPCAT2 gene in RAW264.7 macrophages. ( A , E , I ) The melt curves for TLR4 , CD14, and MD2 amplicons were obtained by reverse transcribing mRNA to cDNA and amplifying cDNA with real-time qPCR. Using R/ggplot2 (method = “gam”, formula = y~s (x, bs = “cs”)), the smooth curves were created from pooled data from all TLR4 , CD14 , and MD2 RT-qPCR experiments (≥3 independent experiments). ( B , F , J ) A control group of RAW264.7 macrophages were transiently transfected with negative siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. After incubation, the cells were lysed for RNA extraction and RT-qPCR. ( C , G , K ) A group of RAW264.7 macrophages was transiently transfected with LPCAT2 siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. ( D , H , L ) LPCAT2 gene expression in the control group is compared with that in the silenced LPCAT2 group. Bars and error bars represent the mean ± standard error of the mean. *— p ≤ 0.05, **— p < 0.01, ***— p < 0.001, ****— p < 0.0001.

Article Snippet: Semi-rough (Rc) LPS— E. coli J5 and Rough (Re) LPS— E. coli K12 D32m4—was purchased from List Biological Laboratories, Eastbourne, UK.

Techniques: Quantitative RT-PCR, Control, Transfection, Infection, Incubation, RNA Extraction, Gene Expression

Journal: Biology

Article Title: Lysophosphatidylcholine Acetyltransferase 2 ( LPCAT2 ) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype

doi: 10.3390/biology13050314

Figure Lengend Snippet: Transcriptional effects of overexpressing LPCAT2 on TLR4 and CD14 gene expression in RAW264.7 macrophages. A control group of RAW264.7 macrophages were transiently transfected with either empty vectors (plasmid vectors with no target sequence) or LPCAT2 vectors (plasmid vectors with LPCAT2 sequence) and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. After incubation, the cells were lysed for RNA extraction and RT-qPCR. ( A – C ) Comparison of LPCAT2 , TLR4, and CD14 gene expression in non-infected and LPS-infected cells. ( D – F ) Comparison of LPCAT2, TLR4 , and CD14 gene expression in the control group and overexpressed LPCAT2 group. Bars and error bars represent the mean ± standard error of the mean. *— p ≤ 0.05, **— p < 0.01, ***— p < 0.001, ****— p < 0.0001. Data were obtained from at least three independent experiments.

Article Snippet: Semi-rough (Rc) LPS— E. coli J5 and Rough (Re) LPS— E. coli K12 D32m4—was purchased from List Biological Laboratories, Eastbourne, UK.

Techniques: Gene Expression, Control, Transfection, Plasmid Preparation, Sequencing, Infection, Incubation, RNA Extraction, Quantitative RT-PCR, Comparison

Journal: Biology

Article Title: Lysophosphatidylcholine Acetyltransferase 2 ( LPCAT2 ) Influences the Gene Expression of the Lipopolysaccharide Receptor Complex in Infected RAW264.7 Macrophages, Depending on the E. coli Lipopolysaccharide Serotype

doi: 10.3390/biology13050314

Figure Lengend Snippet: Transcriptional effects of silencing LPCAT2 gene in RAW264.7 macrophages. ( A , E , I , M ). The melt curves for TNF Alpha , IL6 , IFN Beta , and IP10 amplicons were obtained by reverse transcribing mRNA to cDNA and amplifying cDNA with real-time qPCR. Using R/ggplot2 (method = “gam”, formula = y~s (x, bs = “cs”)), the smooth curves were created from pooled data from all TNF Alpha , IL6 , IFN Beta , and IP10 RT-qPCR experiments (≥3 independent experiments). ( B , F , J , N ) A control group of RAW264.7 macrophages were transiently transfected with negative siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. After incubation, the cells were lysed for RNA extraction and RT-qPCR. ( C , G , K , O ) A group of RAW264.7 macrophages was transiently transfected with LPCAT2 siRNA and infected with either E. coli O111:B4, J5, or K12, D31m4 LPS serotypes; non-infected cells were used as negative controls. ( D , H , L , P ) LPCAT2 gene expression in the control group is compared with that in the silenced LPCAT2 group. Bars and error bars represent the mean ± standard error of the mean. *— p ≤ 0.05, **— p < 0.01, ***— p < 0.001, ****— p < 0.0001.

Article Snippet: Semi-rough (Rc) LPS— E. coli J5 and Rough (Re) LPS— E. coli K12 D32m4—was purchased from List Biological Laboratories, Eastbourne, UK.

Techniques: Quantitative RT-PCR, Control, Transfection, Infection, Incubation, RNA Extraction, Gene Expression

Journal: PLoS ONE

Article Title: Effect of Solar Particle Event Radiation and Hindlimb Suspension on Gastrointestinal Tract Bacterial Translocation and Immune Activation

doi: 10.1371/journal.pone.0044329

Figure Lengend Snippet: Terminal ileum obtained 4 days post irradiation and/or 6 days post hindlimb suspension or from control animals was stained for LPS using a mouse mAb specific for E. coli LPS. A) Ileum from a control treated mouse. B) Ileum from a mouse irradiated with 2 Gy of 70 MeV protons. C) Ileum from a mouse subjected to hindlimb suspension. D) Ileum from a mouse treated with hindlimb suspension and irradiated with 2 Gy of protons. Original magnifications 200×. Tissue from 10 animals was analyzed for each condition.

Article Snippet: Tissue was cut into 6 µm sections in a Leica CM1850 cryostat, fixed in acetone (Thermo Fisher), and stained with rabbit anti-mouse Claudin-3 IgG (Thermo Fisher), goat anti-Lipopolysaccharide IgG (US Biologicals), or a murine monoclonal antibody to Escherichia coli (J5) LPS (Acris Antibodies) and detected with biotinylated anti-rabbit, goat, or mouse IgG (Sigma), streptavidin horseradish-peroxidase (Vector Labs), and AEC substrate (Sigma).

Techniques: Irradiation, Staining

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

doi: 10.1007/s13361-018-1897-y

Figure Lengend Snippet: Agonists were cultured with HEK-Blue hTLR4 cells over a 5-log dose range from 0.1–1000 ng mL−1. W3110 E. coli LPS (red), J5 E. coli LPS (orange), J5 dLPS (pink), J5 dLPS/GBOMP (green), or PHAD (blue) were incubated for 16 hours. Then NF-κB activation was measured by quantification of SEAP in the supernatant. Mean ± SD of duplicate samples and an associated 4-parameter non-linear regression are shown

Article Snippet: Multi-stage MS (MS n ) to confirm structural inferences Intact E. coli J5 LPS from both List Biological Laboratories, Inc. (Campbell, CA) and Sigma Aldrich (St. Louis, MO) were directly infused in a solution composed of 50% 2-propanol and 50% water at a flow rate of 2 μL min −1 into a home-built nESI source of a linear ion trap (linear trapping quadrupole; LTQ) mass spectrometer (Thermo Finnigan, San Jose, CA) with post-production ion funnel optics added for increased ion transmission efficiency.

Techniques: Cell Culture, Incubation, Activation Assay

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

doi: 10.1007/s13361-018-1897-y

Figure Lengend Snippet: Averaged IMS-CID tandem mass spectra of (a) J5 LPS m/z 1071 and (b) J5 dLPS m/z 789 after collision energy ramping. Insets show deprotonated fatty acid product ions’ presence in (a) and absence in (b)

Article Snippet: Multi-stage MS (MS n ) to confirm structural inferences Intact E. coli J5 LPS from both List Biological Laboratories, Inc. (Campbell, CA) and Sigma Aldrich (St. Louis, MO) were directly infused in a solution composed of 50% 2-propanol and 50% water at a flow rate of 2 μL min −1 into a home-built nESI source of a linear ion trap (linear trapping quadrupole; LTQ) mass spectrometer (Thermo Finnigan, San Jose, CA) with post-production ion funnel optics added for increased ion transmission efficiency.

Techniques:

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

doi: 10.1007/s13361-018-1897-y

Figure Lengend Snippet: Zoomed negative mode FT-ICR mass spectrum (R ~ 300,000 FWHM, in absorption mode) after direct infusion of J5 LPS. Eight potential isotopic distribution envelopes can be identified in absorption mode in this 4 m/z window; these are denoted, at the m/z of their respective monoisotopic ions, with blue arrows. (inset) Magnified portion of the spectrum showing fine detail (including the magnitude mode and the proposed overlap between isotopologues from envelopes 2 and 5)

Article Snippet: Multi-stage MS (MS n ) to confirm structural inferences Intact E. coli J5 LPS from both List Biological Laboratories, Inc. (Campbell, CA) and Sigma Aldrich (St. Louis, MO) were directly infused in a solution composed of 50% 2-propanol and 50% water at a flow rate of 2 μL min −1 into a home-built nESI source of a linear ion trap (linear trapping quadrupole; LTQ) mass spectrometer (Thermo Finnigan, San Jose, CA) with post-production ion funnel optics added for increased ion transmission efficiency.

Techniques:

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

doi: 10.1007/s13361-018-1897-y

Figure Lengend Snippet: MS3 CID mass spectra from MS2 product ions representing J5 E. coli lipid A at m/z 1796 (top) and core OS at m/z 1418 (bottom). Similar dissociation phenomena were observed as in MS2 experiments for chemically isolated lipid A and oligosaccharides, indicating feasibility of LPS top down sequencing in this manner

Article Snippet: Multi-stage MS (MS n ) to confirm structural inferences Intact E. coli J5 LPS from both List Biological Laboratories, Inc. (Campbell, CA) and Sigma Aldrich (St. Louis, MO) were directly infused in a solution composed of 50% 2-propanol and 50% water at a flow rate of 2 μL min −1 into a home-built nESI source of a linear ion trap (linear trapping quadrupole; LTQ) mass spectrometer (Thermo Finnigan, San Jose, CA) with post-production ion funnel optics added for increased ion transmission efficiency.

Techniques: Isolation, Sequencing

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

doi: 10.1007/s13361-018-1897-y

Figure Lengend Snippet: Agonists were cultured with HEK-Blue hTLR4 cells over a 5-log dose range from 0.1–1000 ng mL−1. W3110 E. coli LPS (red), J5 E. coli LPS (orange), J5 dLPS (pink), J5 dLPS/GBOMP (green), or PHAD (blue) were incubated for 16 hours. Then NF-κB activation was measured by quantification of SEAP in the supernatant. Mean ± SD of duplicate samples and an associated 4-parameter non-linear regression are shown

Article Snippet: Purified (low protein and nucleic acid content) and lyophilized LPS from E. coli J5 strain was purchased from both List Biological Laboratories, Inc. (Campbell, CA) and Sigma Aldrich (St. Louis, MO).

Techniques: Cell Culture, Incubation, Activation Assay

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

doi: 10.1007/s13361-018-1897-y

Figure Lengend Snippet: Averaged IMS-CID tandem mass spectra of (a) J5 LPS m/z 1071 and (b) J5 dLPS m/z 789 after collision energy ramping. Insets show deprotonated fatty acid product ions’ presence in (a) and absence in (b)

Article Snippet: Purified (low protein and nucleic acid content) and lyophilized LPS from E. coli J5 strain was purchased from both List Biological Laboratories, Inc. (Campbell, CA) and Sigma Aldrich (St. Louis, MO).

Techniques:

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

doi: 10.1007/s13361-018-1897-y

Figure Lengend Snippet: Zoomed negative mode FT-ICR mass spectrum (R ~ 300,000 FWHM, in absorption mode) after direct infusion of J5 LPS. Eight potential isotopic distribution envelopes can be identified in absorption mode in this 4 m/z window; these are denoted, at the m/z of their respective monoisotopic ions, with blue arrows. (inset) Magnified portion of the spectrum showing fine detail (including the magnitude mode and the proposed overlap between isotopologues from envelopes 2 and 5)

Article Snippet: Purified (low protein and nucleic acid content) and lyophilized LPS from E. coli J5 strain was purchased from both List Biological Laboratories, Inc. (Campbell, CA) and Sigma Aldrich (St. Louis, MO).

Techniques:

Journal: Journal of the American Society for Mass Spectrometry

Article Title: Top down tandem mass spectrometric analysis of a chemically modified rough-type lipopolysaccharide vaccine candidate

doi: 10.1007/s13361-018-1897-y

Figure Lengend Snippet: MS3 CID mass spectra from MS2 product ions representing J5 E. coli lipid A at m/z 1796 (top) and core OS at m/z 1418 (bottom). Similar dissociation phenomena were observed as in MS2 experiments for chemically isolated lipid A and oligosaccharides, indicating feasibility of LPS top down sequencing in this manner

Article Snippet: Purified (low protein and nucleic acid content) and lyophilized LPS from E. coli J5 strain was purchased from both List Biological Laboratories, Inc. (Campbell, CA) and Sigma Aldrich (St. Louis, MO).

Techniques: Isolation, Sequencing