Journal: Journal of Inflammation (London, England)

Article Title: Differential expression of nitric oxide synthases in porcine aortic endothelial cells during LPS-induced apoptosis

doi: 10.1186/1476-9255-9-47

Figure Lengend Snippet: Forward and reverse primer sequences, RT-PCR product length and accession number (Acc.No.) in the EMBL database

Article Snippet: The membranes were then incubated overnight at 4°C with a 1:200 dilution of an anti-iNOS (610332, BD Transduction) rabbit polyclonal antibody in PBS-T20 with 3% milk powder or at 4°C overnight with a 1:500 dilution of an anti-eNOS mouse monoclonal antibody (Sa-258, Biomol, Butler Pike, Plymouth Meeting, PA, USA) in Tris Buffered Saline-T20 (TBS-T20 20 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.1% T-20).

Techniques: Sequencing

Journal: Journal of Inflammation (London, England)

Article Title: Differential expression of nitric oxide synthases in porcine aortic endothelial cells during LPS-induced apoptosis

doi: 10.1186/1476-9255-9-47

Figure Lengend Snippet: Relative gene expression of iNOS and eNOS in pAECs under standard culture conditions at different time periods. Relative mRNA data are expressed as delta Ct (HPRT Ct - iNOS or eNOS Ct). The data represent the mean ± SEM (n = 3). Time of culture did not influence iNOS and eNOS mRNA expression.

Article Snippet: The membranes were then incubated overnight at 4°C with a 1:200 dilution of an anti-iNOS (610332, BD Transduction) rabbit polyclonal antibody in PBS-T20 with 3% milk powder or at 4°C overnight with a 1:500 dilution of an anti-eNOS mouse monoclonal antibody (Sa-258, Biomol, Butler Pike, Plymouth Meeting, PA, USA) in Tris Buffered Saline-T20 (TBS-T20 20 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.1% T-20).

Techniques: Gene Expression, Expressing

Journal: Journal of Inflammation (London, England)

Article Title: Differential expression of nitric oxide synthases in porcine aortic endothelial cells during LPS-induced apoptosis

doi: 10.1186/1476-9255-9-47

Figure Lengend Snippet: Relative gene expression of iNOS (A) and eNOS (B) in pAECs treated with LPS (10 μg/ml) at different time periods (n = 3). LPS increased iNOS mRNA expression while eNOS mRNA expression was decreased. Relative mRNA data are expressed as the fold of increase (ΔΔCt method) in respect to the control (CTR = mean ± SEM of all control time points). Error bars represent the range of relative expression. The different letters above the bars indicate significant differences in the various time points (p < 0.05, ANOVA post hoc Tukey’s test).

Article Snippet: The membranes were then incubated overnight at 4°C with a 1:200 dilution of an anti-iNOS (610332, BD Transduction) rabbit polyclonal antibody in PBS-T20 with 3% milk powder or at 4°C overnight with a 1:500 dilution of an anti-eNOS mouse monoclonal antibody (Sa-258, Biomol, Butler Pike, Plymouth Meeting, PA, USA) in Tris Buffered Saline-T20 (TBS-T20 20 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.1% T-20).

Techniques: Gene Expression, Expressing, Control

Journal: Journal of Inflammation (London, England)

Article Title: Differential expression of nitric oxide synthases in porcine aortic endothelial cells during LPS-induced apoptosis

doi: 10.1186/1476-9255-9-47

Figure Lengend Snippet: Expression of eNOS protein in pAECs treated with LPS (10 μg/ml) for different time periods (7, 15, 24 h). A ) LPS induced a significant decrease of eNOS protein expression in respect to the control (CTR = mean ± SEM of all control time points). The data represent the mean ± SEM (n = 3) of the relative protein content (AU = Arbitrary Units). The different letters above the bars indicate significant differences among the various time points (p < 0.05, ANOVA post hoc Tukey’s test, n = 3). B ) Representative Western Blot of eNOS and relative housekeeping β-tubulin were reported.

Article Snippet: The membranes were then incubated overnight at 4°C with a 1:200 dilution of an anti-iNOS (610332, BD Transduction) rabbit polyclonal antibody in PBS-T20 with 3% milk powder or at 4°C overnight with a 1:500 dilution of an anti-eNOS mouse monoclonal antibody (Sa-258, Biomol, Butler Pike, Plymouth Meeting, PA, USA) in Tris Buffered Saline-T20 (TBS-T20 20 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.1% T-20).

Techniques: Expressing, Control, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Differential expression of nitric oxide synthases in porcine aortic endothelial cells during LPS-induced apoptosis

doi: 10.1186/1476-9255-9-47

Figure Lengend Snippet: Representative eNOS immunofluorescent staining on pAEC. Green fluorescence indicates positive immunostaining revealed by FITC, red fluorescence indicates nuclear staining with Propidium Iodide (magnification 400X). A , B ) eNOS protein localisation in standard cultured pAECs; a cytoplasmatic signal evenly diffused was detected. ( C - F ) LPS treatment resulted in a modification of the signal with more intense staining in the perinuclear region.

Article Snippet: The membranes were then incubated overnight at 4°C with a 1:200 dilution of an anti-iNOS (610332, BD Transduction) rabbit polyclonal antibody in PBS-T20 with 3% milk powder or at 4°C overnight with a 1:500 dilution of an anti-eNOS mouse monoclonal antibody (Sa-258, Biomol, Butler Pike, Plymouth Meeting, PA, USA) in Tris Buffered Saline-T20 (TBS-T20 20 mM Tris–HCl, pH 7.4, 500 mM NaCl, 0.1% T-20).

Techniques: Staining, Fluorescence, Immunostaining, Cell Culture, Modification

Journal: Nature

Article Title: A metabolomics pipeline for mechanistic interrogation of the gut microbiome

doi: 10.1038/s41586-021-03707-9

Figure Lengend Snippet: a, Top panel: An example decision tree from a forest that can differentiate Bacteroidetes vs. bacteria from the other four represented phyla with > 97% accuracy. For each decision node, phylum-level elevation and reduction based on metabolite levels are shown (relative fold change to bacterial media controls, log2 transformed). Actinobacteria (n = 20), Bacteroidetes (n = 57), Firmicutes (n = 83), Fusobacteria (n = 3), and Proteobacteria (n = 10). Dashed line: metabolite threshold. Box: median, 25th, and 75th percentile; whiskers: Tukey’s method. Bottom panel: The 10 most important features differentiating the five tested phyla. Data are shown as median metabolite log2 fold-change for each phylum; metabolites and phyla are ordered by Ward linkage distance. b, Representative growth curves from two independent experiments, each with n = 3 biological replicates for a subset of Bacteroides spp. using modified Salyer’s Minimal Medium (SMM) with indicated nitrogen source. Legend colors for sole nitrogen source are maintained for panels b-d. c, Representative growth curves of one experiment with n = 5 biological replicates for 60 Bacteroidetes using modified SMM with indicated nitrogen sources. d, Growth curves of wild-type and mutant Bt grown in defined minimal media with either cysteine (top) (one experiment, n = 3 biological replicates) or sodium sulfide (Na2S, bottom) as sole reduced sulfur sources (one experiment, n = 3 biological replicates). e, Amino acid production and consumption levels in gnotobiotic mice mono-associated with Bacteroides thetaiotaomicron (Bt) (one experiment, n = 5 mice). Box: median, 25th, and 75th percentile; whiskers: Tukey’s method). Numeric labels in b and c correspond to the following: 1: B. acidifaciens DSMZ 15896, 2: B. caccae ATCC 43185, 3: B. caccae BEI HM-728, 4: B. cellulosilyticus BEI HM-726, 5: B. cellulosilyticus DSMZ 14838, 6: B. coprophilus DSMZ 18228, 7: B. dorei BEI HM-29, 8: B. dorei BEI HM-717, 9: B. dorei BEI HM-718, 10: B. dorei BEI HM-719, 11: B. dorei DSMZ 17855, 12: B. eggerthii ATCC 27754, 13: B. eggerthii DSMZ 20697, 14: B. finegoldii BEI HM-727, 15: B. finegoldii DSMZ 17565, 16: B. fragilis BEI HM-20, 17: B. fragilis BEI HM-710, 18: B. fragilis BEI HM-711, 19: B. fragilis BEI HM-714, 20: B. fragilis NCTC 9343, 21: B. intestinalis DSMZ 17393, 22: B. ovatus ATCC 8483, 23: B. ovatus BEI HM-222, 24: B. pectinophilus ATCC 43243, 25: B. plebeius DSMZ 17135, 26: B. salyersiae BEI HM-725, 27: B. sp. BEI HM-18, 28: B. sp. BEI HM-189, 29: B. sp. BEI HM-19, 30: B. sp. BEI HM-22, 31: B. sp. BEI HM-23, 32: B. sp. BEI HM-258, 33: B. sp. BEI HM-27, 34: B. sp. BEI HM-28, 35: B. sp. BEI HM-58, 36: B. stercoris ATCC 43183, 37: B. stercoris BEI HM-1036, 38: B. thetaiotaomicron 3730, 39: B. thetaiotaomicron 3731, 40: B. thetaiotaomicron 633, 41: B. thetaiotaomicron 7330, 42: B. thetaiotaomicron 7853, 43: B. thetaiotaomicron 8702, 44: B. thetaiotaomicron 8713, 45: B. thetaiotaomicron 8736, 46: B. thetaiotaomicron 940, 47: B. thetaiotaomicron VPI 5482, 48: B. thetaiotaomicron wh302, 49: B. thetaiotaomicron wh305, 50: B. uniformis ATCC 8492, 51: B. vulgatus ATCC 8482, 52: B. vulgatus BEI HM-720, 53: B. xylanisolvens DSMZ 18836, 54: P. distasonis ATCC 8503, 55: P. distasonis BEI HM-169, 56: P. johnsonii BEI HM-731, 57: P. johnsonii DSMZ 18315, 58: P. merdae ATCC 43184, 59: P. merdae BEI HM-729, 60: P. merdae BEI HM-730.

Article Snippet: BEI HM-19, 30: B. sp. BEI HM-22, 31: B. sp. BEI HM-23, 32: B. sp. BEI HM-258, 33: B. sp. BEI HM-27, 34: B. sp. BEI HM-28, 35: B. sp. BEI HM-58, 36: B. stercoris ATCC 43183, 37: B. stercoris BEI HM-1036, 38: B. thetaiotaomicron 3730, 39: B. thetaiotaomicron 3731, 40: B. thetaiotaomicron 633, 41: B. thetaiotaomicron 7330, 42: B. thetaiotaomicron 7853, 43: B. thetaiotaomicron 8702, 44: B. thetaiotaomicron 8713, 45: B. thetaiotaomicron 8736, 46: B. thetaiotaomicron 940, 47: B. thetaiotaomicron VPI 5482, 48: B. thetaiotaomicron wh302, 49: B. thetaiotaomicron wh305, 50: B. uniformis ATCC 8492, 51: B. vulgatus ATCC 8482, 52: B. vulgatus BEI HM-720, 53: B. xylanisolvens DSMZ 18836, 54: P. distasonis ATCC 8503, 55: P. distasonis BEI HM-169, 56: P. johnsonii BEI HM-731, 57: P. johnsonii DSMZ 18315, 58: P. merdae ATCC 43184, 59: P. merdae BEI HM-729, 60: P. merdae BEI HM-730. fig ft0 fig @position="anchor" mode=article f4 fig mode="anchored" f5 Extended Data Fig. 9, fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 Metabolic contribution by individual gut microbes in a multi-species community. a, Alpha-ketoglutaric acid levels in feces of mice mono-colonized with Anaerostipes sp. BEI HM-220.

Techniques: Bacteria, Transformation Assay, Modification, Mutagenesis

Journal: Nature

Article Title: A metabolomics pipeline for mechanistic interrogation of the gut microbiome

doi: 10.1038/s41586-021-03707-9

Figure Lengend Snippet: a, Alpha-ketoglutaric acid levels in feces of mice mono-colonized with Anaerostipes sp. BEI HM-220. Mean ± s.e.m. of two independent experiments, each with n = 4 mice (germ-free) or n = 5 or 7 mice (Anaerostipes mono-colonized). b, Left panel: MDMs were associated with specific bacterial phyla leveraging both in vivo and in vitro metabolomic data. Right panel: Number of mega-medium grown bacterial strains by phylum that produce MDMs identified in the cecal contents of mice colonized with Bacteroides thetaiotaomicron (Bt, n = 5), or Clostridium sporogenes (Cs, n = 3), or a six-member community (n = 3). Number of strains that produce at least one of these metabolites in vitro by phylum: Bacteroidetes: n = 52, Firmicutes: n = 60, Proteobacteria: n = 8, Actinobacteria: n = 16, and Fusobacteria: n = 3. Each metabolite shown was significantly produced both in vitro and in vivo (≥ 4-fold, corrected P < 0.05). Uniquely detected (non-coeluting) metabolites are shown (Supplementary Table 9) .c, Spearman correlation between metabolomic profiles (standardized and scaled, log2-transformed, fold-change data) of individual Bt- or Cs-mono-associated host biofluids (cecal contents, feces, serum, or urine) and individual bacterial culture (158 mega-medium grown). Colored dots: Spearman’s rho values calculated by comparing metabolomic profiles of individual bacterial culture vs. individual biofluid of either Bt- or Cs-mono-associated mice. Black dots: Spearman’s rho calculated using metabolomic profiles of Bt or Cs, the same strains used for mono-association in mice. d, Venn diagram of overlapping metabolites that are significantly produced (≥ 4-fold, corrected P < 0.05) in culture and in the cecum of colonized mice. e, Principal component analysis (PCA) separates metabolomic profiles of identified metabolites by sample type in each colonization state. P values on metabolomic profile comparisons between different sample types of the same colonization state were determined using PERMANOVA: six-member community (P = 0.073), and all other colonization states (P = 0.001). f, PCA separates metabolomic profiles of identified metabolites by colonization states. P values on metabolomic profile comparisons between different colonization states of the same sample type were determined using PERMANOVA: P = 0.001 for all four sample types. g, h, Example chemical structures of significantly produced metabolites (≥ 4-fold, corrected P < 0.05) in serum (g) or urine (h) by each colonization state corresponding to Fig. 4b. P values in a, b, d, g, h: two-tailed t-test with Benjamini-Hochberg correction for multiple comparisons.

Article Snippet: BEI HM-19, 30: B. sp. BEI HM-22, 31: B. sp. BEI HM-23, 32: B. sp. BEI HM-258, 33: B. sp. BEI HM-27, 34: B. sp. BEI HM-28, 35: B. sp. BEI HM-58, 36: B. stercoris ATCC 43183, 37: B. stercoris BEI HM-1036, 38: B. thetaiotaomicron 3730, 39: B. thetaiotaomicron 3731, 40: B. thetaiotaomicron 633, 41: B. thetaiotaomicron 7330, 42: B. thetaiotaomicron 7853, 43: B. thetaiotaomicron 8702, 44: B. thetaiotaomicron 8713, 45: B. thetaiotaomicron 8736, 46: B. thetaiotaomicron 940, 47: B. thetaiotaomicron VPI 5482, 48: B. thetaiotaomicron wh302, 49: B. thetaiotaomicron wh305, 50: B. uniformis ATCC 8492, 51: B. vulgatus ATCC 8482, 52: B. vulgatus BEI HM-720, 53: B. xylanisolvens DSMZ 18836, 54: P. distasonis ATCC 8503, 55: P. distasonis BEI HM-169, 56: P. johnsonii BEI HM-731, 57: P. johnsonii DSMZ 18315, 58: P. merdae ATCC 43184, 59: P. merdae BEI HM-729, 60: P. merdae BEI HM-730. fig ft0 fig @position="anchor" mode=article f4 fig mode="anchored" f5 Extended Data Fig. 9, fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window caption a7 caption a8 Metabolic contribution by individual gut microbes in a multi-species community. a, Alpha-ketoglutaric acid levels in feces of mice mono-colonized with Anaerostipes sp. BEI HM-220.

Techniques: In Vivo, In Vitro, Produced, Transformation Assay, Two Tailed Test