parental strain e coli k 12 mg1655  (ATCC)

Journal: eLife

Article Title: Nociceptor neurons control pollution-mediated neutrophilic asthma

doi: 10.7554/eLife.101988

Figure Lengend Snippet: ( A – C ) Male and female C57BL/6 mice (6–10 weeks of age) were sensitized via intraperitoneal injection with an emulsion of ovalbumin (OVA; 200 µg/dose) and aluminum hydroxide (1 mg/dose) on days 0 and 7. On days 14–16, mice were challenged intranasally with OVA (50 µg/dose), either alone or in combination with fine particulate matter (FPM; 20 µg/dose). Bronchoalveolar lavage fluid was collected, and jugular-nodose complex neurons were cultured on day 17 for 24 hr before being loaded with the calcium indicator Fura-2AM. Cells were sequentially stimulated with the TRPA1 agonist AITC (successively to 10 µM at 60–90 s, 30 µM at 90–120 s, 100 µM at 120–150 s) and then with KCl (40 mM at 420–435 s). Calcium flux was continuously monitored throughout the experiment. The amplitude of AITC responses was measured by calculating the ratio of peak F340/F380 fluorescence after stimulation to the baseline F340/F380 fluorescence measured 30 s prior to stimulation. Data are plots as the per dish average of AITC and KCl responsive neurons and show that AITC (10 µM) responses were higher in JNC neurons from OVA-FPM-exposed mice when compared to vehicle or OVA alone ( C ). Data in are presented as means ± SEM ( B–C ). N are as follows: ( B ) n=35 neurons (control group), 19 neurons (OVA group), and 38 neurons (OVA + FPM group), ( C ) n=5 dishes totaling 35 neurons (control group), 8 dishes totaling 42 neurons (OVA group), and 10 dishes totaling 76 neurons (OVA + FPM group). p-Values were determined by nested one-way ANOVA with post hoc Bonferroni’s. p-Values are shown in the figure.

Article Snippet: Parental strains C57BL/6 (# 000664), Dta fl/fl (# 010527, # 009669), tdTomato fl/fl (# 007914), Trpv1 cre/cre (# 017769), and Scn10a cre/cre (# 036564) were purchased from The Jackson Laboratory.

Techniques: Injection, Emulsion, Cell Culture, Fluorescence, Control

Journal: eLife

Article Title: Nociceptor neurons control pollution-mediated neutrophilic asthma

doi: 10.7554/eLife.101988

Figure Lengend Snippet: ( A – B ) Male and female C57BL/6 mice (6–10 weeks of age) were sensitized intraperitoneally with ovalbumin (OVA; 200 µg/dose in 200 µl) and aluminum hydroxide (1 mg/dose in 200 µl) on days 0 and 7. On days 14–16, mice were challenged intranasally with OVA (50 µg/dose in 50 µl) alone or with fine particulate matter (FPM; 20 µg/dose in 50 µl). On day 16, 30 min after the final challenge, mice received intranasal QX-314 (5 nmol/dose in 50 µl). Bronchoalveolar lavage fluid (BALF) was collected on day 17 and analyzed by flow cytometry. Compared with naïve or OVA-exposed mice, those co-challenged with OVA+FPM showed increased BALF neutrophils ( A ). QX-314 treatment normalized these levels, while BALF eosinophil levels remained comparable ( B ). ( C – E ) Male and female littermate control (TRPV1 WT ) and nociceptor-ablated (TRPV1 DTA ) mice (6–10 weeks of age) were sensitized and challenged under the same OVA±FPM protocol (days 0, 7, and 14–16). BALF or lungs were collected on day 17 and assessed by flow cytometry. Compared with naïve or OVA-exposed mice, OVA+FPM co-challenged mice exhibited higher BALF neutrophils ( C ) and lung γδ T cells ( E ). Nociceptor ablation protected against these increases ( C, E ), while BALF eosinophil levels remained comparable ( D ). Data are shown as mean ± SEM ( A – E ). Experiments were replicated twice, and animals pooled ( A–E ). N are as follows: ( A–B ) control (n=6), OVA (n=7), OVA-FPM (n=12), OVA-FPM+QX-314 (n=10), ( C–D ) TRPV1 WT + control (n=9), TRPV1 WT + OVA (n=13), TRPV1 WT + OVA-FPM (n=18), TRPV1 DTA + OVA-FPM (n=19), ( E ) TRPV1 WT + control (n=3), TRPV1 WT + OVA (n=3), TRPV1 WT + OVA-FPM (n=4), TRPV1 DTA + OVA-FPM (n=5). p-Values were determined by a one-way ANOVA with post hoc Tukey’s ( A–E ). p-Values are shown in the figure.

Article Snippet: Parental strains C57BL/6 (# 000664), Dta fl/fl (# 010527, # 009669), tdTomato fl/fl (# 007914), Trpv1 cre/cre (# 017769), and Scn10a cre/cre (# 036564) were purchased from The Jackson Laboratory.

Techniques: Flow Cytometry, Control

Journal: eLife

Article Title: Nociceptor neurons control pollution-mediated neutrophilic asthma

doi: 10.7554/eLife.101988

Figure Lengend Snippet: ( A–B ) Male and female littermate control (TRPV1 WT ) and nociceptor-ablated (TRPV1 DTA ) mice (6–10 weeks of age) were sensitized and challenged under the same ovalbumin (OVA)±fine particulate matter (FPM) protocol (days 0, 7, and 14–16). Bronchoalveolar lavage fluid (BALF) was collected on day 17 and assessed by multiplex array and enzyme-linked immunosorbent assay (ELISA). Compared with naïve or OVA-alone groups, OVA+FPM co-challenged mice exhibited levels of TNFα and artemin. Notably, ablating nociceptors prevented these increases. ( C ) In silico analysis of the GSE124312 dataset . The heatmap displays transcript expression levels for the pan neural-crest lineage transcription factor ( Prdm12 ), voltage-gated sodium channels ( Scn9a, Scn10a ), jugular subset markers ( Wfdc2, Mrgprd, Osmr, Sstr2, Nefh, Trpm8 ), peptidergic neuron markers ( Trpa1, Trpv1, Calca, Tac1, Gfra3 ), and the pan placodal lineage marker ( Phox2b ). Gfra3 expression is enriched in the peptidergic neuron cluster labeled JG4. Experimental details and cell clustering are described by . ( D ) In silico analysis of GSE192987 showing co-expression of Gfra3 with Trpa1 and other inflammatory markers. Data are visualized as row z-scores in a heatmap or via UMAPs (TPTT>1). Experimental details and cell clustering are described by . ( E–G ) Alveolar macrophages (3×10 5 cells/well) from naïve male and female C57BL/6 mice were cultured overnight and then stimulated with vehicle (DMSO) or FPM (100 µg/ml). RNA was extracted 1 and 4 hr post-stimulation, and Artn expression was assessed using quantitative PCR (qPCR). FPM exposure increased Artn transcript levels at both 1 and 4 hr ( F, G ). ( H–J ) Naïve mice jugular-nodose-complex neurons were harvested, pooled, and cultured overnight with either vehicle or artemin (100 ng/ml). Cells were sequentially stimulated with AITC (TRPA1 agonist; 300 µM at 240–270 s), capsaicin (TRPV1 agonist; 300 nM at 320–335 s), and KCl (40 mM at 720–735 s). The percentage of AITC-responsive neurons (among all KCl-responsive cells) was normalized to vehicle-treated controls for each batch of experiments. Artemin-treated neurons showed increased responsiveness to AITC, while responses to capsaicin and KCl were unchanged ( I–J ). Data are presented as means ± SEM ( A–B, F–G, J ), heatmap displaying the z-score of DESeq2 normalized counts ( C ), tSNE plots ( D ), schematics ( E, H ), means ± 95% CI of maximum Fura-2AM (F/F 0 ) fluorescence ( I ). N are as follows: ( A ) TRPV1 WT + control (n=2), TRPV1 WT + OVA (n=3) TRPV1 WT + OVA-FPM (n=3), TRPV1 DTA + OVA-FPM (n=8), ( B ) TRPV1 WT + OVA (n=6) TRPV1 WT + OVA-FPM (n=8), TRPV1 DTA + OVA-FPM (n=14), ( F ) n=2/time point, ( G ) n=8/group, ( I ) vehicle (n=107 neurons), artemin (n=122 neurons); ( J ) n=4/group. p-Values were determined by a one-way ANOVA with post hoc Tukey’s ( A, B ) or unpaired Student’s t-test ( G, J ). p-Values are shown in the figure.

Article Snippet: Parental strains C57BL/6 (# 000664), Dta fl/fl (# 010527, # 009669), tdTomato fl/fl (# 007914), Trpv1 cre/cre (# 017769), and Scn10a cre/cre (# 036564) were purchased from The Jackson Laboratory.

Techniques: Control, Multiplex Assay, Enzyme-linked Immunosorbent Assay, In Silico, Expressing, Marker, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Antibiotics

Article Title: In Vitro and In Vivo Evaluation of Nitroxoline as an Effective Antimicrobial Alternative to Poultry Production

doi: 10.3390/antibiotics15010062

Figure Lengend Snippet: NTX-resistant mutant selection and stability. ( a ) Spontaneous mutation frequency of NTX resistance in wild-type S. enterica and E. coli strains was measured at 2×, 4×, and 8× MIC (8, 16, and 32 mg/L) after 48 h incubation at 37 °C. The resistance of colonies was supported by their ability to grow on NTX-containing media at the indicated concentrations. The frequency of resistance was determined by dividing the number of resistant mutants by the total number of cells determined by using dilutions of the overnight culture on agar media. Data are presented as mean ± Standard Error of the Mean (SEM) from two technical replicates ( n = 2). No mutations were observed at the concentration of 4× or 8× MIC (16 and 32 µg/mL, respectively). ( b ) MIC fold changes in selected mutant relative to their parental strains from ( a ). Parent–mutant pairs are indicated by vertical dotted lines. ( c ) Serial passage induction of the resistance to NTX against reference (hollow circle) and wild-type (solid star) E. coli and S. enterica . The y axis is the MIC-fold change in the tested isolates. Data are presented as mean ± SEM from two biological replicates ( n = 2). ( d ) Stability of NTX resistance mutations in two mutants from E. coli ATCC 25922 (Mut ATCC-D13 and Mut ATCC-D14) and two mutants from E. coli DSM 103263 (Mut DSM-D8 and Mut DSM-D14) in the absence of NTX. Mutant stability was quantified as the percentage of mutants in the original bacterial population, calculated by dividing the viable mutant cell count resistant to NTX by the total population and multiplying by a factor of 100. Three biological and two technical replicates were performed for each strain. Data are presented as mean ± SEM ( n = 3 biological replicates, each with two technical replicates).

Article Snippet: Parental strains E. coli ATCC 25922 and E. coli DSM 103263 were patched on drug-free medium only as controls.

Techniques: Mutagenesis, Selection, Incubation, Concentration Assay, Cell Characterization