r36a  (ATCC)

Journal: Journal of Microbiology and Biotechnology

Article Title: Serotype-Dependent Inhibition of Streptococcus pneumoniae Growth by Short-Chain Fatty Acids

doi: 10.4014/jmb.2309.09003

Figure Lengend Snippet: S. pneumoniae ( A ) D39, ( B ) R36A, and ( C ) R6, were cultured in media with or without various doses of SCFAs. The OD 600 value was measured at 0, 1, 3, 6, 9, 12, or 24 h. ( D ) At maximum growth time point of each NT, the susceptibility is calculated as OD 600 value of 100 mM SCFAs treated group divided by maximal OD 600 value of each NT. Data shown are representative of three different experiments and indicate the mean value ± standard deviation of triplicate samples. Statistical significance was analyzed with Student’s t -test compared to NT or encapsulated strain (*, P < 0.05). NaA, sodium acetate; NaP, sodium propionate; NaB, sodium butyrate; NT, nontreatment.

Article Snippet: S. pneumoniae serotype 3 (ATCC 6303), TIGR4 (ATCC BAA-334), 19F (ATCC 49619), and R36A (ATCC 27336) were purchased from American Type Culture Collection (USA).

Techniques: Cell Culture, Standard Deviation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pneumococcal surface protein A plays a major role in Streptococcus pneumoniae -induced immunosuppression

doi: 10.4049/jimmunol.1502709

Figure Lengend Snippet: (A) PC expression by R36A or R36Apc- by flow cytometry (“control”, R36Apc- stained with FITC-anti IgG2a only [no primary anti-PC Ab]). BALB/c mice (7 per group) were immunized i.v. with 50 μg cOVA in alum: (B) with or without 2×108 CFU R36A or R36Apc-, (C) R36A (grown in standard THB medium) or CDM-grown R36A, or (D) R36A, sonicated R36A or sonicated R36Apc-. All mice were boosted i.v. with 50 μg cOVA + alum without bacteria, on d14. Serum titers of cOVA-specific and PC-specific IgG were measured by ELISA. Significance * p≤0.05

Article Snippet: SDS PAGE 2μl of R36A cbp- supernatant (equivalent to 6×10 7 CFU R36A) or 1×10 7 WT or trypsin-treated R36A, was mixed with NuPAGE sample reducing agent (10x) and NuPage LDS sample buffer (4x) (Life Technologies, Carlsbad, CA) to a final concentration of 1x in 25ul total sample volume.

Techniques: Expressing, Flow Cytometry, Control, Staining, Sonication, Bacteria, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pneumococcal surface protein A plays a major role in Streptococcus pneumoniae -induced immunosuppression

doi: 10.4049/jimmunol.1502709

Figure Lengend Snippet: (A) cOVA-specific Tg T cells from DO11.10 mice were adoptively transferred into BALB/c mice and 1 d later immunized i.v. with PBS only or 50 μg cOVA in alum with or without 2 × 108 CFU R36A or R36Apc- (3-5 mice/group). On d 8 following immunization gated CD4+ DO11.10 TCR+ Tg T cells from spleen cell suspensions were analyzed for GC Tfh cell phenotype (GL7hi PD1hi). (B) BALB/c mice (4-5/group) were immunized i.v. with PBS only or 50 μg NP-cOVA with or without 2 × 108 CFU R36A or R36Apc-. On d 14 post-immunization, NP-specific B cells (B220+ CD11C− NP+) were analyzed for isotype-switched GC NP+ B cells (IgD−/low PNA+) (C) Quantitation of NP-specific ASC from spleen and BM in response to NP-cOVA alone or with R36A or R36Apc- on d 14 post-immunization (4-5 mice/group). Significance * p≤0.05

Article Snippet: SDS PAGE 2μl of R36A cbp- supernatant (equivalent to 6×10 7 CFU R36A) or 1×10 7 WT or trypsin-treated R36A, was mixed with NuPAGE sample reducing agent (10x) and NuPage LDS sample buffer (4x) (Life Technologies, Carlsbad, CA) to a final concentration of 1x in 25ul total sample volume.

Techniques: Quantitation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pneumococcal surface protein A plays a major role in Streptococcus pneumoniae -induced immunosuppression

doi: 10.4049/jimmunol.1502709

Figure Lengend Snippet: BALB/c mice (7/group) were immunized i.v. with 50μg cOVA in alum with or without: (A) 50μg PB-BSA or NP-BSA (as control) or 2 × 108 CFU R36A, (B) 2 × 108 CFU WT R36A or periodate-treated R36A, (D) 4×108 CFU E. rhusiopathiae or 2×108 CFU N. meningitidis, or (E) 2×108 CFU R36A or 5×108 CFU H. influenzae.. All mice were boosted i.v. with 50 μg cOVA + alum without bacteria, on d14. Serum titers of cOVA-specific IgG were measured by ELISA. (C) PC expression by R36A or R36A (periodate) by flow cytometry (“control”, R36A stained with FITC-anti IgG2a only [no primary anti-PC Ab]). Significance * p≤0.05.

Article Snippet: SDS PAGE 2μl of R36A cbp- supernatant (equivalent to 6×10 7 CFU R36A) or 1×10 7 WT or trypsin-treated R36A, was mixed with NuPAGE sample reducing agent (10x) and NuPage LDS sample buffer (4x) (Life Technologies, Carlsbad, CA) to a final concentration of 1x in 25ul total sample volume.

Techniques: Control, Bacteria, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pneumococcal surface protein A plays a major role in Streptococcus pneumoniae -induced immunosuppression

doi: 10.4049/jimmunol.1502709

Figure Lengend Snippet: BALB/c mice (7 per group) were immunized i.v. with 50 μg cOVA in alum with or without 2 × 108 CFU R36A or R36Acbp-. All mice were boosted i.v. with 50 μg cOVA in alum without bacteria, on d14. Serum titers of (A) cOVA-specific, (C) PC-specific and (D) PspA-specific IgG were measured by ELISA. (B) PC and PspA expression by R36A or R36Acbp- by flow cytometry (“control”, R36A stained with FITC-anti IgG2a only [no primary anti-PspA or anti-PC Ab]). Significance * p≤0.05.

Article Snippet: SDS PAGE 2μl of R36A cbp- supernatant (equivalent to 6×10 7 CFU R36A) or 1×10 7 WT or trypsin-treated R36A, was mixed with NuPAGE sample reducing agent (10x) and NuPage LDS sample buffer (4x) (Life Technologies, Carlsbad, CA) to a final concentration of 1x in 25ul total sample volume.

Techniques: Bacteria, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry, Control, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pneumococcal surface protein A plays a major role in Streptococcus pneumoniae -induced immunosuppression

doi: 10.4049/jimmunol.1502709

Figure Lengend Snippet: BALB/c mice (7 per group) were immunized i.v. with 50 μg cOVA in alum with or without (A) 2×108 CFU R36A or supernatant of choline chloride-treated R36A (R36Acbp- sup.) equivalent to 2×108 CFU R36A or (B) 2×108 CFU R36A or trypsin treated R36A [R36A (trypsin)]. All mice were boosted i.v. with 50 μg cOVA in alum without bacteria, on d 14. Serum titers of cOVA-specific IgG were measured by ELISA. (C) Lysates from R36A cells, R36A (trypsin) and R36Acbp- sup., electrophoresed on SDS-PAGE. Significance * p≤0.05.

Article Snippet: SDS PAGE 2μl of R36A cbp- supernatant (equivalent to 6×10 7 CFU R36A) or 1×10 7 WT or trypsin-treated R36A, was mixed with NuPAGE sample reducing agent (10x) and NuPage LDS sample buffer (4x) (Life Technologies, Carlsbad, CA) to a final concentration of 1x in 25ul total sample volume.

Techniques: Bacteria, Enzyme-linked Immunosorbent Assay, SDS Page

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Pneumococcal surface protein A plays a major role in Streptococcus pneumoniae -induced immunosuppression

doi: 10.4049/jimmunol.1502709

Figure Lengend Snippet: BALB/c mice (7 per group) were immunized i.v. with 50 μg cOVA in alum with or without (A) 2×108 CFU R36A or T4R or T4R mutants for the respective CBPs listed in the graph. (B) 2×108 CFU T4R or T4R PspA mutant (ΔPspA). (C) 2×108 intact R36A, 50 μg recombinant, truncated PspA, or 50 μg recombinant full-length LytA. All mice were boosted i.v. with 50 μg cOVA in alum without bacteria, on d 14. Serum titers of cOVA-specific IgG were measured by ELISA. Significance * p<0.05.

Article Snippet: SDS PAGE 2μl of R36A cbp- supernatant (equivalent to 6×10 7 CFU R36A) or 1×10 7 WT or trypsin-treated R36A, was mixed with NuPAGE sample reducing agent (10x) and NuPage LDS sample buffer (4x) (Life Technologies, Carlsbad, CA) to a final concentration of 1x in 25ul total sample volume.

Techniques: Mutagenesis, Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

doi: 10.7554/eLife.65651

Figure Lengend Snippet: Apparent disassembly or exchange rate constants (min −1 ) and ParB F /ParA F ratio from fits of TIRFM wash and FRAP experiments. The apparent dissociation (or FRAP) rate constants ( k off ) were obtained for individual time-trajectories by single-exponential curve fitting (except ** where the rate of the faster decay, (68 ± 0.5%) of a double-exponential fit is shown), and the mean and SEM for the set of independent experiments are shown (except * where standard deviation among non-independent repeats within an experiment is shown). (N is the number of separate experiments, with total number of binding/wash cycles for repeated data collection in parenthesis.) ParA:ParB ratios were calculated from the final phase of the individual association time-trajectories (except *** where it was based on the beginning part of the washing phase in the presence of ParB F 1-42 R36A ), and the mean and SEM for the set of independent experiments are shown in italics. N.D., not done.

Article Snippet: ParB F 1-42 and ParB F 1-42 R36A were synthesized de novo (Genscript).

Techniques: Standard Deviation, Binding Assay

Journal: eLife

Article Title: CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

doi: 10.7554/eLife.65651

Figure Lengend Snippet: ATPase fit parameters. ATPase measurements were performed with ParA F (1 μM) and different mutants of ParB F , 60 μg/ml EcoRI-digested pBR322 DNA plus Scram- or parS F -DNA fragment and CTP or CDP, as indicated in the column headings. Assays were repeated ‘ N ’ times, each data set of an assay was fit after subtraction of background measured without ParA F to a modified Hill equation: v − v 0 = (v max [B] n ) / (K A n + [B] n ), and the mean and standard error of the mean (SEM) of the fit parameters for the N measurements are shown. For [B] on the x-axis, total ParB F concentration was used instead of free ParB F concentration due to technical issues in estimating the free ParB F concentration and the meanings of K A and the cooperativity factor (n) here differ from those in the standard adaptation of the Hill equation. v max is the maximum stimulated ParA F ATPase turnover rate, K A is the apparent total concentration of ParB F necessary for half maximum stimulation, and n is the apparent cooperativity coefficient.

Article Snippet: ParB F 1-42 and ParB F 1-42 R36A were synthesized de novo (Genscript).

Techniques: Modification, Concentration Assay

Journal: eLife

Article Title: CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

doi: 10.7554/eLife.65651

Figure Lengend Snippet: ( A ) ParB F 1-42 R36A -mCherry (10 μM) and ParA F -eGFP (1 μM) preincubated with ATPγS were infused into the nsDNA-carpeted flow cell and then ( B ) washed with buffer containing ATPγS. ( C ) The washing experiment of B was repeated with buffer containing ATPγS and ParB F 1-42 R36A -mCherry (10 μM). ( D ) ParA F -ATPase activity was measured in the presence of EcoRI-digested pBR322 DNA (60 μg/ml) as a function of ParB F 1-42 R36A concentration. See legend and and for additional details.

Article Snippet: ParB F 1-42 and ParB F 1-42 R36A were synthesized de novo (Genscript).

Techniques: Activity Assay, Concentration Assay