Journal: MedComm

Article Title: Cholesterol 25‐Hydroxylase Enhances Myeloid‐Derived Suppressor Cell (MDSC) Immunosuppression via the Stimulator of Interferon Genes (STING)‐Tank‐Binding Kinase 1 (TBK1)‐Receptor‐Interacting Protein Kinase 3 (RIPK3) Pathway in Colorectal Cancer

doi: 10.1002/mco2.70411

Figure Lengend Snippet: Regulation of MDSCs by 25HC is dependent on the cGAS–STING pathway. (A) Venn plot analysis of RNA transcript sequencing results of WT MDSCs treated with 25HC, either alone or in combination with MC38‐TCM, as well as Ch25h f/f Lyz2 Cre(±) MDSCs (with Ch25h f/f MDSCs serving as the control). Acas: acetoacetyl‐coenzyme A synthetase; Cyp51: cytochrome P450 family 51; Dhcr24: 24‐dehydrocholesterol reductase; Fdps: farnesyl diphosphate synthase; Gnas: guanine nucleotide‐binding protein, alpha stimulating; Idi1: isopentenyl‐diphosphate delta isomerase 1; Sqle: squalene epoxidase. (B) cGAS–STING scores were calculated by aggregating gene sets from the GOBP_CGAS_STING_SIGNALING_PATHWAY.v2024.1.Hs pathway in the MSigDB database ( https://www.gsea‐msigdb.org/gsea/msigdb ) using GSVA. The CH25H and cGAS–STING scores were analyzed using Spearman correlation analysis and visualized with scatter plots. (C) Changes in cGAS and STING, as well as ARG1 protein levels in WT MDSCs, were detected via Western blotting (WB) following 25HC treatment (using DMSO as the control). (D) The phosphorylation activation level of STING and the protein level changes of cGAS and ARG1 were assessed after CH25H‐specific knockdown and subsequent treatment with 25HC in MDSCs from both the mock group (induced by RPMI1640 medium) and the MC38‐TCM treatment group (using Ch25h f/f MDSCs as the control). p/t: phosphorylated protein/total protein. (E) The changes in ARG1 expression in Ch25h f/f Lyz2 Cre(±) MDSCs (using Ch25h f/f MDSCs as the control) were evaluated after treatment with C176 (1 µM; using the same volume of DMSO as the control condition) for 6 h. (F, G) WB and flow cytometry were employed to detect ARG1 expression in MDSCs from Tmem173 ‐KO mice (using WT MDSCs as the control). (H) WB analysis of ARG1 expression levels in Tmem173 ‐KO MDSCs (using WT MDSCs as the control) induced by the combination of 25HC and MC38‐TCM treatment. (I) A functional inhibition assay was conducted to assess T cell activity after 48 h of coculture of Tmem173 ‐KO MDSCs with CD8 + T cells, using WT MDSCs as the control. Comparisons with controls are indicated, where ns denotes no significant difference, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: For C176 treatment, following the injection of MC38 cells for 8 days, mice received intraperitoneal injections of C176 (Selleck, Catalogue No. S6575) at a volume of 200 μL (11.5 mg/kg, 5% DMSO + 40% PEG300 + 5% Tween80 [VETEC, Catalogue No. V900507] + 50% sterile saline) or control vector every 2 days, and were euthanized after 20 days.

Techniques: Sequencing, Control, Binding Assay, Western Blot, Phospho-proteomics, Activation Assay, Knockdown, Expressing, Flow Cytometry, Functional Assay, Inhibition, Activity Assay

Journal: MedComm

Article Title: Cholesterol 25‐Hydroxylase Enhances Myeloid‐Derived Suppressor Cell (MDSC) Immunosuppression via the Stimulator of Interferon Genes (STING)‐Tank‐Binding Kinase 1 (TBK1)‐Receptor‐Interacting Protein Kinase 3 (RIPK3) Pathway in Colorectal Cancer

doi: 10.1002/mco2.70411

Figure Lengend Snippet: CH25H deletion inhibits ARG1 expression by activating the TBK1–RIPK3 complex. (A) WB analysis shows the phosphorylation levels and total protein levels of TBK1 and RIPK3 in WT MDSCs treated with 25HC (using DMSO as the control condition). (B) The activation levels of TBK1 and RIPK3 phosphorylation, as well as their total levels, are assessed following CH25H‐specific knockdown and subsequent treatment with 25HC in MDSCs from both the mock group and the MC38‐TCM treatment group (using Ch25h f/f MDSCs as the control). (C) Similar assessments of TBK1 and RIPK3 phosphorylation and total levels are conducted after CH25H‐specific knockdown and C176 stimulation in MDSCs from the mock and MC38‐TCM treatment groups (using Ch25h f/f MDSCs as the control). (D) The total and phosphorylated expression levels of TBK1 and RIPK3 are compared between WT and Tmem173 ‐KO MDSCs, using WT MDSCs as the control. (E) WB analysis reveals the protein phosphorylation activation levels and total expression of TBK1 and RIPK3 in Tmem173 ‐KO MDSCs induced by the combination of 25HC and MC38‐TCM treatment, using WT MDSCs as the control. (F) WB demonstrates the phosphorylation levels and total protein levels of TBK1, alongside ARG1 expression in WT MDSCs treated with GSK8612 at a concentration of 10 µM for 24 h (using DMSO as the control condition). (G) The alterations in ARG1 and TBK1 expression in Ch25h f/f Lyz2 Cre(±) MDSCs (using Ch25h f/f MDSCs as the control) following a similar treatment with GSK8612 (10 µM) for 24 h. (H) CoIP assays demonstrate the interactions between TBK1 and RIPK3 proteins in Ch25h f/f Lyz2 Cre(±) MDSCs compared to Ch25h f/f MDSCs. (I) Flow cytometry assesses ARG1 expression in Ch25h f/f Lyz2 Cre(±) MDSCs, with Ch25h f/f MDSCs serving as a control, after 24 h of treatment with GSK872 (20 µM), either alone or in combination with MC38‐TCM ( n = 3). (J) Flow cytometry assays evaluate the functional inhibition of CD8 + T cells following 48 h of coculture with MDSCs treated with GSK872, with or without MC38‐TCM (using Ch25h f/f MDSCs as the control; n = 3). (K) Flow cytometry assays assess the inhibitory effects on CD8 + T cell proliferation after 72 h of coculture with MDSCs treated with GSK872, both in the presence and absence of MC38‐TCM (using Ch25h f/f MDSCs as the control; n = 3). Statistical comparisons are made against controls, with ns indicating no significant difference, * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: For C176 treatment, following the injection of MC38 cells for 8 days, mice received intraperitoneal injections of C176 (Selleck, Catalogue No. S6575) at a volume of 200 μL (11.5 mg/kg, 5% DMSO + 40% PEG300 + 5% Tween80 [VETEC, Catalogue No. V900507] + 50% sterile saline) or control vector every 2 days, and were euthanized after 20 days.

Techniques: Expressing, Phospho-proteomics, Control, Activation Assay, Knockdown, Concentration Assay, Flow Cytometry, Functional Assay, Inhibition

Journal: MedComm

Article Title: Cholesterol 25‐Hydroxylase Enhances Myeloid‐Derived Suppressor Cell (MDSC) Immunosuppression via the Stimulator of Interferon Genes (STING)‐Tank‐Binding Kinase 1 (TBK1)‐Receptor‐Interacting Protein Kinase 3 (RIPK3) Pathway in Colorectal Cancer

doi: 10.1002/mco2.70411

Figure Lengend Snippet: CH25H‐deficient MDSCs can reverse their antitumor effects following stimulation with 25HC and C176. Panels A–F present physical images of solid tumor volume (A), tumor dynamic growth curves (B), and mass (C), as well as ARG1 expression in tumor MDSCs (D), the percentage of CD8 + TILs in CD45 + cells (E), and the percentage of IFNγ + or GzmB + CD8 + TILs (F) in Ch25h f/f Lyz2 Cre(±) mice treated with or without 25HC ( n = 4). Panels G–L depict physical drawings of solid tumor volume (G), tumor dynamic growth curves (H), and mass (I), along with ARG1 expression in tumor MDSCs (J), the percentage of CD8 + TILs in CD45 + cells (K), and the percentage of IFNγ + or GzmB + CD8 + TILs (L) in Ch25h f/f Lyz2 Cre(±) mice treated with or without C176 ( n = 5). Statistical comparisons indicate no significant difference (ns) compared to controls, with significance levels of * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: For C176 treatment, following the injection of MC38 cells for 8 days, mice received intraperitoneal injections of C176 (Selleck, Catalogue No. S6575) at a volume of 200 μL (11.5 mg/kg, 5% DMSO + 40% PEG300 + 5% Tween80 [VETEC, Catalogue No. V900507] + 50% sterile saline) or control vector every 2 days, and were euthanized after 20 days.

Techniques: Expressing

Journal: BMC neuroscience

Article Title: Delta opioid receptors affect acoustic features of song during vocal learning in zebra finches.

doi: 10.1186/s12868-025-00927-x

Figure Lengend Snippet: Fig. 1 Quantitative analysis of spectral and temporal features of motifs sung by naltrindole-treated and control birds from 80dph to 120dph. The bars represent means, the error bars represent standard deviations and dots represent mean feature value per bird. (a) Birds treated with naltrindole during the sensitive period of learning tended to have shorter syllables than their control siblings, although these differences were not statistically significant. There were no significant differences between (b) intersyllable intervals of control and treated birds. There was a significant effect of treatment with δ-OR antagonist naltrindole on (c) pitch, (d) mean frequency and (e) frequency modulation, all of which were significantly lower compared with the same measures from vehicle-treated siblings. Results of Bonferroni post-hoc tests for these measures are shown above the bars representing different ages

Article Snippet: The selective non-peptidic δ-OR antagonist naltrindole (cat: 7040, CAS number: 111469-81-9; Tocris Bioscience, Ellisville, MO, USA) was dissolved in 0.9% saline to obtain a stock solution of 2.2mM (1 mg/ml).

Techniques: Control

Journal: BMC neuroscience

Article Title: Delta opioid receptors affect acoustic features of song during vocal learning in zebra finches.

doi: 10.1186/s12868-025-00927-x

Figure Lengend Snippet: Fig. 2 (a) Sonogram representing an exemplar of an FM syllable. These are complex syllables with intricate frequency traces. (b) Sonogram repre senting a harmonic stack syllable which is simpler than FM syllables and consists of frequency traces arranged in a stack above the fundamental frequency. Both these types of syllables were used to measure the motif complexity of the naltrindole-treated birds and their control siblings. Fig ures show average number of syllable types produced by the (c) control and (d) treated birds. Each point in the scatter plot represents data for one bird for the number of FM or H-stack type syllables per motif. (c) There was a significantly greater number of FM syllables in the songs of controls, whereas (d) songs of birds administered the δ-OR antagonist naltrindole tended to have a greater number of harmonic stacks

Article Snippet: The selective non-peptidic δ-OR antagonist naltrindole (cat: 7040, CAS number: 111469-81-9; Tocris Bioscience, Ellisville, MO, USA) was dissolved in 0.9% saline to obtain a stock solution of 2.2mM (1 mg/ml).

Techniques: Control, Produced

Journal: BMC neuroscience

Article Title: Delta opioid receptors affect acoustic features of song during vocal learning in zebra finches.

doi: 10.1186/s12868-025-00927-x

Figure Lengend Snippet: Fig. 3 Analysis of spectral and temporal features of harmonic-stack (Hstck) and FM syllables. Bars represent mean values; error bars represent standard error and dots represent feature value per syllable. (a) Frequency modulated syllables were produced at a higher pitch by controls than naltrindole- treated birds. For H-Stack syllables, values of pitch were slightly lower in songs of control birds. (b) The value for mean frequency obtained after analyzing both types of syllables was higher in songs of control birds, compared to those sung by their naltrindole-treated siblings (c) Frequency modulation was not significantly different for the H-stack syllables in the two groups of birds, but was lower in naltrindole-treated versus control birds for FM syllables. (d) No significant differences were observed between Wiener entropy values for control and treated birds for both types of syllables. (e) The frequency- modulated syllables produced by controls were significantly longer than those produced by treated birds. No such temporal difference was observed for the harmonic stack syllables sung by control and naltrindole-treated siblings

Article Snippet: The selective non-peptidic δ-OR antagonist naltrindole (cat: 7040, CAS number: 111469-81-9; Tocris Bioscience, Ellisville, MO, USA) was dissolved in 0.9% saline to obtain a stock solution of 2.2mM (1 mg/ml).

Techniques: Produced, Control

Journal: BMC neuroscience

Article Title: Delta opioid receptors affect acoustic features of song during vocal learning in zebra finches.

doi: 10.1186/s12868-025-00927-x

Figure Lengend Snippet: Fig. 4 Comparison of syllable order between control and naltrindole-treated birds. Bars graphs represent mean values and error bars represent standard deviation. The vertical scatter plots for these graphs represent the value of mean similarity and stereotypy values per bird in all graphs. Values for (a) linearity, (b) consistency and (c) stereotypy were higher for controls than for naltrindole-treated birds, with the largest difference observed in linearity

Article Snippet: The selective non-peptidic δ-OR antagonist naltrindole (cat: 7040, CAS number: 111469-81-9; Tocris Bioscience, Ellisville, MO, USA) was dissolved in 0.9% saline to obtain a stock solution of 2.2mM (1 mg/ml).

Techniques: Comparison, Control, Standard Deviation

Journal: BMC neuroscience

Article Title: Delta opioid receptors affect acoustic features of song during vocal learning in zebra finches.

doi: 10.1186/s12868-025-00927-x

Figure Lengend Snippet: Fig. 6 Quantification of DARPP-32-positive neurons in the basal ganglia song control nucleus Area X. Well-labelled DARPP-32-positive cells were pres ent in Area X and LMANshell of both control (a, c) and treated (b, d) birds, respectively. Insets in these figures show DARPP-32-positive neurons at high magnification (Scale bar, 100 μm; for insets, 10 μm). For Fig. 6e and f, bar graphs represent mean values, and error bars represent standard deviation; the scatter plot represents the average number of DARPP-32-positive cells per bird. (e) Administration of the δ-OR antagonist naltrindole for a 10-day period during the sensitive period of learning resulted in a significant increase in the number of DARPP32-positive medium spiny neurons in Area X. (f) Although the number of DARPP32-positive cells were also higher in the LMANshell of treated birds, this difference was not significant

Article Snippet: The selective non-peptidic δ-OR antagonist naltrindole (cat: 7040, CAS number: 111469-81-9; Tocris Bioscience, Ellisville, MO, USA) was dissolved in 0.9% saline to obtain a stock solution of 2.2mM (1 mg/ml).

Techniques: Control, Standard Deviation

Journal: BMC neuroscience

Article Title: Delta opioid receptors affect acoustic features of song during vocal learning in zebra finches.

doi: 10.1186/s12868-025-00927-x

Figure Lengend Snippet: Fig. 7 (a) Single bands were obtained in western blots performed using zebra finch brain lysate for PSD-95 at 95kD, Synaptotagmin at 65kD and DARPP- 32 at 32kD using specific antibodies (anti-PSD-95, ab9708, Abcam, USA; anti-synaptotagmin, MAB5200, MERCK Millipore; anti-DARPP32, ab40801, Abcam, USA). The synaptic markers were used to label synapses in (b) control and (c) treated birds, respectively. Insets show a magnified view of the synaptic contacts (yellow). (d) There was a significant increase in the number of possibly excitatory synapses within the song control nucleus Area X of treated birds during development, suggesting that altering δ-OR signaling can cause long term changes in synaptic connectivity within the basal ganglia. Scale bar, 10 μm. Bar graphs represent mean values and error bars represent the standard deviation. Each dot in the scatter plot represents average puncta values per field per bird. (e) Comparisons of the number of synapses between siblings showed that a higher number of synapses were observed in Area X of the naltrindole-treated birds versus controls in each set

Article Snippet: The selective non-peptidic δ-OR antagonist naltrindole (cat: 7040, CAS number: 111469-81-9; Tocris Bioscience, Ellisville, MO, USA) was dissolved in 0.9% saline to obtain a stock solution of 2.2mM (1 mg/ml).

Techniques: Western Blot, Control, Standard Deviation

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: Visualization of import of multiple DNA molecules within one DNA uptake location. Fluorescein (F) and Cy3 labelled DNA was either added for 30 min to separate C. jejuni 81–176 suspensions (( A ), n ≥ 5) or in parallel to one bacterial competent culture (( B , C ), n = 4) and the fraction of cells with F and/or Cy3 fluorescent foci were analyzed. In ( A ) the fraction of active cells relative to the total number of bacteria is shown, in ( B ) the fraction of cells with fluorescent foci relative to the overall fraction of competent cells is depicted. Green bars, cells with only fluorescein labelled DNA foci; orange bars, cells with only Cy3 labelled DNA foci; yellow bar, cells with both fluorescein and Cy3 DNA in one/or two single focus/foci; green/orange hatched bar, cells with at least two separate foci, with either fluorescein or Cy3 DNA. ( C ), overlay image of differential phase contrast (DIC), Cy3 and F channel. Green, fluorescein focus; orange, Cy3 focus.

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Bacteria

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: The fraction of competent C. jejuni and of DNA uptake locations per cell increased in time of contact with fluorescent DNA. Competent C. jejuni 81–176 were exposed to fluorescent DNA for different time periods and the fraction of cells with active DNA uptake was monitored in comparison to 10 min DNA uptake in H. pylori J99 ( A ). Proportion of cells with distinct number of DNA uptake locations ( B ). In ( B ), fraction of cells relative to total amount of competent cells are depicted. Mean and standard deviations were derived from five independent experiments.

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Comparison, Derivative Assay

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: The median amount of DNA within single foci of C. jejuni was constant over time and ~4-fold less than in H. pylori . C. jejuni 81–176 and H. pylori J99 were incubated with the same batch of fluorescent genomic C. jejuni DNA labelled with Cy3. Uptake was followed in time for C. jejuni and compared to 10 min uptake in H. pylori . ( A ) Analysis of DNA foci in C. jejuni (red; light to dark corresponds to incubation times) and in H. pylori (blue), sorted according to fluorescence intensity. ( B ) ( C. jejuni ) and ( C ) ( H. pylori ) distribution of fluorescence intensities as boxplots; the boxplot length corresponds to the interquartile range (IQR; 50%) of data, the horizontal bar indicates the median value; whiskers represent 1.5 × IQR or the maximum/minimum value of the dataset; dots, outliers. Upper panel, merged DIC and Cy3-channel example images of C. jejuni and H. pylori cells after import of DNA. Note that Cy3 exposure times were 25 ms for H. pylori and 100 ms for C. jejuni . Scale bar of 2 µm. One representative experiment (out of three) is shown.

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Incubation, Fluorescence

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: Biofilm formation is independent of active DNA transport or natural transformation in C. jejuni . Upon growth in microtiter plates for 72 h and suitable washing of unbound bacteria, crystal violet staining indicated the intensity of biofilm formation, measured as absorbance at 595 nm. C. jejuni 81–176 (wt) and its isogenic mutants Δ pilQ , Δ comE , Δ comEC and the respective complemented strains were tested, using Pseudomonas aeruginosa as reference for a typical biofilm forming bacterium. Horizontal bar, mean of ≥3 independent experiments; n.s., not significant; *, p < 0.05; ***, p < 0.001.

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Transformation Assay, Bacteria, Staining

Journal: Biomolecules

Article Title: Cj0683 Is a Competence Protein Essential for Efficient Initialization of DNA Uptake in Campylobacter jejuni

doi: 10.3390/biom13030514

Figure Lengend Snippet: Cj0683 plays a pivotal role for DNA uptake into the periplasm of C. jejuni . DNA uptake ( A ) and transformation rate ( B ) in 81–176 (wt) and the mutant strains Δ cj0683, Δ cj0683-compl and Δ cj0683 Δ comE . ( A ) Cells were either incubated with fluorescein- (green bars, except for Δ cj0683 Δ comE ) or Cy3-labelled (orange bars) genomic DNA of 81–176 for 30 min. ( B ) Transformation rate was determined using unlabeled genomic DNA containing streptomycin resistance. Error bars indicate standard deviation ( n ≥ 4).

Article Snippet: C. jejuni 81–176 [ ] from −80 °C stock cultures (MAST Group Ltd., Bootle, UK) was grown on Columbia blood agar plates supplemented with 5% defibrinated sheep blood (ColbA, Oxoid, Thermo Fisher Scientific Inc., Waltham, MA, USA) under microaerobic conditions (5% O 2 , 10% CO 2 , rest N 2 ; Binder, Tuttlingen, Germany).

Techniques: Transformation Assay, Mutagenesis, Incubation, Standard Deviation

Journal: BMC Microbiology

Article Title: Roles of RpoN in the resistance of Campylobacter jejuni under various stress conditions

doi: 10.1186/1471-2180-11-207

Figure Lengend Snippet: Growth of the rpoN mutant under different aeration conditions . The C. jejuni strains were microaerobically cultured in MH broth at 42°C with shaking at 180 rpm (A) and without shaking (B). At the described time intervals, the optical density at 600 nm was measured, and viable cells were counted in static culture condition (without shaking) by plating serially-diluted C. jejuni cultures on MH agar plates. The results are the mean ± standard deviation of three independent experiments. ***: P < 0.001; the significance of results was statistically analyzed by two-way ANOVA analysis of variance with Bonferroni's post-tests at a 95% confidence interval using Prism software (version 5.01; GraphPad Software Inc., USA).

Article Snippet: We thank Dr. Qijing Zhang (Iowa State University, USA) for providing C. jejuni 81-176.

Techniques: Mutagenesis, Cell Culture, Standard Deviation, Software

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: Bacterial strains and plasmids used in this study.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Mutagenesis, Plasmid Preparation, Homologous Recombination

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: (A) A scheme for the PLP production pathway (right box) in C. jejuni in relation to Pse biosynthesis (left box) is illustrated based on in silico pathway analysis performed using PATRIC ( http://patricbrc.vbi.vt.edu/portal/portal/patric/Home ). (B) The pdxA mutant produced no PLP. The C. jejuni 81–176 WT, pdxA mutant, and the complemented strains were grown in 10ml of MH broth to an OD 600 of 0.60. The suspensions were then homogenized, serially diluted, and subjected to ELISA to quantify the amounts of PLP (μg 10 ml −1 ). The data show the mean +/− standard deviations from three independent assays.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: In Silico, Mutagenesis, Produced, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: (A) The pdxA mutant shows less glycosylation of FlaA. SDS-PAGE and western blotting were conducted to detect the C. jejuni FlaA protein. Crude extracts and subcellular (cytoplasmic and membrane) fractions were extracted from C. jejuni and visualized using CBB staining in an SDS-polyacrylamide gel (left panel). Western blot analyses were simultaneously performed to detect the FlaA protein (arrow, right panel). (B) The pdxA mutant shows reduced Pse production. The left panel shows an extracted ion chromatogram at m / z 441.0–461.0 obtained through SIM of DMB-labeled Pse from the WT and pdxA mutant strains (arrowed). The extracted ion chromatogram of blank sample (fresh MH broth) was simultaneously subjected to confirm the absence of Pse. AA, peak area in arbitrary units. Each ion signal is expressed as a relative percentage of the WT-derived sample (set to 100%) from two independent tests (right panel). MS n data were shown in Fig. S1, S2, S3. (C) The disruption of the pdxA gene impairs motility of C. jejuni . The WT, pdxA mutant, pdxA -complemented ( pdxA −/+), and flaA mutant (flaA-) strains were spotted and incubated onto 0.4% soft agar. Scale bars represent 3 mm. The motility of pdxA mutant was also assayed in the supplementation of 10 mg l −1 of PLP (pdxA− + PLP). (D) The pdxA mutant is aflagellated. Electron micrographs of the C. jejuni WT, pdxA mutant with or without supplementation of PLP (10 mg l −1 ), pdxA - complemented strains. The scale bars represent 1 μm.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Mutagenesis, Glycoproteomics, SDS Page, Western Blot, Membrane, Staining, Labeling, Derivative Assay, Disruption, Incubation

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: Representative metabolites that are altered between the C. jejuni WT and pdxA mutant strains.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Mutagenesis

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: (A) Growth curves of C. jejuni 81–176 WT, pdxA−, and the complemented mutant strains in MH broth not supplemented (left panel) or supplemented (right panel) with PLP (10 mg l −1 ). (B) Intracellular ATP levels of C. jejuni 81–176 WT, pdxA−, and the complemented mutant strains. ATP contents of four serial dilutions of the bacteria (shown as CFU 100 μl −1 ) under investigation were measured. The results are shown as means ± SD of data from triplicate wells of a representative experiment. (C) Focused dynamics of the C. jejuni TCA-cycle pathway. The pathway, the relative mean concentrations of the related metabolites in the WT (blue bars) and the pdxA mutant (red bars) strains, and the genes associated with the enzymatic conversion of each metabolite were illustrated with the PATRIC pathway analysis program.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Mutagenesis, Bacteria

Journal: PLoS ONE

Article Title: Campylobacter jejuni pdxA Affects Flagellum-Mediated Motility to Alter Host Colonization

doi: 10.1371/journal.pone.0070418

Figure Lengend Snippet: (A) INT407 cells were infected for 1 h with the C. jejuni WT, pdxA−, pdxA−/+, and flaA− strains. The number of cell-adherent bacteria was measured by counting the plates after washing three times with PBS. (B) ERK1/2 activation upon infection. Western blotting was performed to detect the levels of phosphorylated and total ERK1/2 in the lysates from infected cells. (C) IL-8 production in INT407 cells was measured at 4 h and 16 h p.i. via ELISA. The data are presented in sections A and C as the mean values ± standard deviations from samples run in duplicate in at least three experiments. (D) Disruption of the pdxA gene reduces the colonization of the chicken cecum by C. jejuni . Groups of 14-day-old chickens (n = 10 per group) were orally inoculated with approximately 3×10 7 CFU of WT or pdxA mutant C. jejuni . At 1 week and 4 weeks p.i. , the ceca were aseptically removed from the infected animals (n = 5 for each time point) and homogenized. Serial dilutions of the suspensions were plated on mCCDA agar to count CFU numbers. The closed diamonds and open circles represent the numbers of WT and pdxA mutant CFUs recovered from the animals, respectively.

Article Snippet: C. jejuni strain 81–176 was grown using routine methods in Mueller-Hinton (MH) broth or on MH agar (Becton-Dickinson, Franklin Lakes, NJ, USA) at 37°C for 24 h in a humidified CO 2 AnaeroPack-Microaero gas system (Mitsubishi Gas Chemicals, Tokyo, Japan).

Techniques: Infection, Bacteria, Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Disruption, Mutagenesis