Journal: Microbiome

Article Title: Antimicrobial peptides modulate lung injury by altering the intestinal microbiota

doi: 10.1186/s40168-023-01673-0

Figure Lengend Snippet: Oxygen exposure reduces intestinal antimicrobial peptide expression. A Neonatal C57BL/6 J mice were exposed to normoxia or hyperoxia from the 3 rd -14. th day of life ( n = 4 litters with 5–7 neonatal mice/litter per exposure group). FiO , fraction of inspired oxygen. SPF, specific-pathogen-free. B Representative photomicrographs of the distal lung sections of 14-day-old mice. C Hyperoxia exposure is associated with alterations in lung morphology and function. Data are shown as mean ± SEM, with significance testing by a two-tailed t -test. D Volcano plot of ileal gene expression array showing gene expression altered by hyperoxia exposure. E Heatmap showing genes regulated by hyperoxia exposure. F Principal components analysis showing differential clustering of normoxia and hyperoxia exposed ileal genes. PC, principal component. G Ileal antimicrobial peptide expression is decreased in hyperoxia-exposure mice. H Community diversity of the adherent and luminal ileal bacterial microbiome is not significantly altered by hyperoxia exposure. I The relative abundance of an operational taxonomic unit (OTU 002) that aligns to the genus Staphylococcus increases after hyperoxia exposure, as do OTUs aligning to Corynebacterium (OTU 124) and Romboutsia (OTU 013). Data are shown as mean ± SEM, with significance testing by a two-tailed t -test. J Principal coordinates analysis of Bray–Curtis dissimilarity shows global alterations in community composition in hyperoxia-exposed mice. Significance testing by permutational ANOVA (PERMANOVA), with equivocal dispersion confirmed by permutational multivariate analysis of dispersion (PERMDISP). PC, principal component. K Loading plot of principal components analysis of Hellinger transformed Euclidian distances showing the contribution of specific genera to the global community composition. Schematic in ( A ) was generated using BioRender. See also Figures S , S and S

Article Snippet: Intestinal organoids were generated and incubated in a hyperoxia chamber (StemCell Technologies) at 95% oxygen for 24 h. After the hyperoxia was completed, the supernatant was removed, and the organoids and Matrigel were collected in 0.5 mL cell recovery media (Corning) per well and placed on ice for 40 min.

Techniques: Expressing, Two Tailed Test, Dispersion, Transformation Assay, Generated

Journal: Microbiome

Article Title: Antimicrobial peptides modulate lung injury by altering the intestinal microbiota

doi: 10.1186/s40168-023-01673-0

Figure Lengend Snippet: Intestinal lysozyme supplementation reduces hyperoxia-induced lung injury. A Neonatal C57BL/6NCrl mice randomized to either every other day exposure to lysozyme by gastric gavage or their littermate controls were then exposed to normoxia or hyperoxia from the 3 rd -14. th day of life ( n = 4 litters with 5–7 neonatal mice/litter per exposure group). FiO 2 , fraction of inspired oxygen. PBS, phosphate-buffered saline (vehicle). SPF, specific-pathogen-free. B Representative photomicrographs of the distal lung sections of 14-day-old mice. C Lysozyme exposure ameliorates hyperoxia-induced disruptions in lung morphology and function. Data are shown as mean ± SEM, with significance testing by two-way ANOVA. D Volcano plot of ileal RNAseq showing gene expression altered by lysozyme exposure. E Heatmap showing genes regulated by lysozyme exposure. F Principal components analysis showing differential clustering of ileal genes in lysozyme-exposed mice. PC, principal component. G Ileal antimicrobial peptide expression is altered in lysozyme-exposed mice. H The community diversity of the adherent and luminal ileal bacterial microbiome is not significantly altered by lysozyme exposure. I The hyperoxia-induced increase in the relative abundance of operational taxonomic unit 014 ( Staphylococcus ) is ameliorated by lysozyme exposure. Multiple other genera are increased in lysozyme and hyperoxia-exposed mice. Data are shown as mean ± SEM, with significance testing by two-way ANOVA. J Principal coordinates analysis of Bray–Curtis dissimilarity shows global alterations in community composition in lysozyme-exposed mice. Significance testing by permutational ANOVA (PERMANOVA), with equivocal dispersion confirmed by permutational multivariate analysis of dispersion (PERMDISP). PC, principal component. K Loading plot of a principal components analysis of a Hellinger transformed Euclidian distance showing global community composition significantly altered in lysozyme-exposed mice. The schematic in ( A ) was generated using BioRender. See also Figure S

Article Snippet: Intestinal organoids were generated and incubated in a hyperoxia chamber (StemCell Technologies) at 95% oxygen for 24 h. After the hyperoxia was completed, the supernatant was removed, and the organoids and Matrigel were collected in 0.5 mL cell recovery media (Corning) per well and placed on ice for 40 min.

Techniques: Saline, Expressing, Dispersion, Transformation Assay, Generated

Journal: Microbiome

Article Title: Antimicrobial peptides modulate lung injury by altering the intestinal microbiota

doi: 10.1186/s40168-023-01673-0

Figure Lengend Snippet: Lysozyme exposure alters the lung transcriptome. A Volcano plot showing hyperoxia alters gene expression in vehicle-exposed controls. B Volcano plot showing lysozyme exposure alters gene expression in the lung. C Heatmap showing differentially expressed genes in vehicle-exposed controls. D Heatmap showing similarly expressed genes between all groups. E Heatmap showing differentially expressed genes in lysozyme-exposed mice. F Major pathways altered in mice only exposed to normoxia or hyperoxia. G Major pathways altered in lysozyme-exposed mice

Article Snippet: Intestinal organoids were generated and incubated in a hyperoxia chamber (StemCell Technologies) at 95% oxygen for 24 h. After the hyperoxia was completed, the supernatant was removed, and the organoids and Matrigel were collected in 0.5 mL cell recovery media (Corning) per well and placed on ice for 40 min.

Techniques: Expressing

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Bronchoalveolar lavage (BAL) fluid analysis determined numbers of neutrophils for inflammation and total protein concentration for vascular permeability (n = 3/group for air and 48 h O2, n = 6/group for 72h O2), and (B) activity of lactate dehydrogenase (LDH) for cytotoxicity (n = 3/group for air, n = 4–5/group for O2). (C) Hyperoxia susceptibility determined by body weight loss was indicated as percent body weight change at the end of 72 h hyperoxia or air exposure (day 18) compared to the onset of the diet (day 1, n = 3/group for air, n = 6/group for O2). Data presented as group mean±SE. Two-way ANOVA used for all statistical analyses. *, P < 0.05 vs. genotype- and diet-matched air controls. +, P < 0.05 vs. diet- and exposure-matched Nrf2+/+ mice. §, P < 0.05 vs genotype- and exposure-matched AIN group.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Protein Concentration, Permeability, Activity Assay

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Heat map from hierarchical clustering analysis depicts expression profiles of SFN-responded genes in Nrf2+/+ after 72 h hyperoxia (O2) exposure (n = 1,187, P < 0.01 with moderated t-test). Heat map for the same genes in Nrf2−/− mice were shown for comparison. Color bar indicates average expression intensity (n = 3/group) normalized to Nrf2+/+-PBS-Air group. (B) Top canonical pathways of lung genes significantly altered by SFN in air-exposed Nrf2+/+ (black bars) and in air-exposed Nrf2−/− mice (grey bars). (C) Mitochondrial oxidative phosphorylation complex is illustrated with genes (in red) that were induced by SFN treatment in Nrf2+/+ mice exposed to normoxia (room air). (D) Top diseases and bio-functions of SFN-responsive lung genes in Nrf2+/+ mice included energy metabolism such as fatty acid beta-oxidation. (E) Lung genes significantly reduced by SFN in Nrf2+/+ mice were involved in the network of organismal injury and abnormality (scores 32–41), in which key molecules such as TNF receptor associated factor 1 (Traf1) and multiple mitogen-activated protein kinase (MAPK) cascade enzymes (e.g., Map3k8) were predicted to play central roles with NF-κB. Analysis was done by Ingenuity Pathway Analysis software.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Expressing, Comparison, Phospho-proteomics, Software

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Pathway analysis for hyperoxia-responsive genes in PBS-received Nrf2+/+ mice (n= 7162 genes, P < 0.01, Moderated t-test) demonstrated p53 as a key upstream regulator for the hyperoxia-altered lung genes, which may sequentially modulate other signal transducers. (B) In Nrf2−/− mice that received PBS, O2 altered genes (n = 4,799, P < 0.01) involved predominantly in IL-17A signaling pathway, which may lead to severe neutrophil infiltration. (C) Nrf2-dependently modulated genes during hyperoxia (n = 816, P < 0.01) such as Selp and Fcgr2b may contribute to the differential lung edema between Nrf2+/+ and Nrf2−/− mice given PBS.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques:

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Top canonical pathways of Nrf2-dependently changed gene transcripts with PBS (top, gray bars) or SFN (bottom, black bars) pretreatment in response to hyperoxia. (B) Profile analysis classified Nrf2-dependently regulated genes by similar expression patterns. (C) Pathway analysis for SFN-responsive genes in hyperoxia-exposed Nrf2−/− mice (n= 533, P < 0.01) depicted that genes altered by SFN only in these mice (e.g., Sele, Itga5, Lif, Flnb) may stimulate cellular movement and interaction by activating cell spreading, attachment, and homing, through which SFN may exert Nrf2-independent responses against hyperoxia in Nrf2−/− mice. Analyses were done using Ingenuity Pathway Analysis and GeneSpring software.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Expressing, Software

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: Mitochondrial genome copy numbers determined by droplet digital PCR (ddPCR).

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Digital PCR

Journal: Toxicology and applied pharmacology

Article Title: Sulforaphane enriched transcriptome of lung mitochondrial energy metabolism and provided pulmonary injury protection via Nrf2 in mice

doi: 10.1016/j.taap.2018.12.004

Figure Lengend Snippet: (A) Immunohistochemical localization of voltage-dependent anion-selective channel 1 (VDAC1)/porin, a mitochondrial membrane potential marker, in lung tissue sections. Brown dots indicate VDAC1-positive cells. Representative light photomicrographs are shown (n = 3–4/group). VDAC1 localization at baseline lung (PBS/Air) is indicated by small arrows. Thick black arrows indicate areas with increased VDAC1 expression compared to genotype-matched PBS/Air. White arrows indicate areas with decreased VDAC1 compared to genotype-matched PBS/Air. SFN = sulforaphane. O2 = Hyperoxia. AV, alveoli; BR, bronchi; BV, blood vessel; PA, pulmonary artery; TB, terminal bronchiole. Bar = 100 μm. (B) Aliquots of lung cytosolic proteins were subjected for Western blotting using specific antibodies. Representative images from multiple analyses of pooled proteins (n = 3/antibody) presented. ATP5A = ATP synthase subunit alpha, mitochondrial. MT-CO1 = mitochondrially encoded cytochrome c oxidase subunit 1. SDH8 = succinate dehydrogenase subunit B. NDUFB8 = NADH dehydrogenase (Ubiquinone) 1 beta subcomplex, 8. VDAC1 = Voltage-dependent anion-selective channel 1. PPLA=Cardiac phospholamban. kDa = kilodalton. Scanned band images were quantitated by densitometry. Data presented as group mean ± SE. Two-way ANOVA used for all statistical analyses. *, P < 0.05 vs. genotype- and pretreatment-matched air controls. +, P < 0.05 vs. pretreatment- and exposure-matched Nrf2+/+ mice. §, P < 0.05 vs. genotype- and exposure-matched PBS group. (C) Aliquots of pooled lung nuclear protein (5 μg) were incubated with an end-labeled oligonucleotide probe containing antioxidant response element (ARE) consensus sequence, and gel shift analysis determined total ARE binding. Nuclear proteins (5 μg) from PBS/hyperoxia-Nrf2−/− mice were run as a negative control. Nuclear proteins were subjected for Western blot analysis using Nrf2-specific antibody and images were quantified. *, P < 0.05 vs. pretreatment-matched air controls. §, P < 0.05 vs. PBS/hyperoxia group. (D) Aliquots of pooled lung nuclear protein (5 μg) were incubated with an end-labeled oligonucleotide probe containing NF-κB consensus sequence, and gel shift analysis determined total NF-κB binding. Two shifted bands (arrow heads) indicate total DNA-NF-κB complex. Specific activity for p65 NF-κB subunit was quantified using a transcription factor ELISA. Nuclear proteins from PBS/hyperoxia-Nrf2−/− mice were used for reaction with cold probes (20 pmol addition of oligonucleotide) and for no antibody control to verify the reaction specificity. *, P < 0.05 vs. genotype- and pretreatment-matched air controls. +, P < 0.05 vs. pretreament- and exposure-matched Nrf2+/+ mice. §, P < 0.05 vs. genotype- and exposure-matched PBS group.

Article Snippet: Mr. Herman Price for coordinating hyperoxia exposures at the NIEHS Inhalation Facility under contract to Alion Science and Technology, Inc. Microarray analysis was performed at the NIEHS Microarray Core, and Ms. Carolyn Favaro and Ms. Isabel Lea in the National Toxicology Program submitted array data to GEO and NIEHS CEBS.

Techniques: Immunohistochemical staining, Membrane, Marker, Expressing, Western Blot, Incubation, Labeling, Sequencing, Gel Shift, Binding Assay, Negative Control, Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in pharmacology

Article Title: Deficiency of endothelial FGFR1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice.

doi: 10.3389/fphar.2022.1039103

Figure Lengend Snippet: FIGURE 1 Hyperoxia disrupted angiogenesis and alveologenesis and resulted in respiratory dysfunction inneonatal mice. (A) Approach to develop a mouse model of BPD. Mouse pups were exposed to room air (21% O2) or hyperoxia (80% O2) from the day of birth (P0) to postnatal days (P)14. After measuring the respiratory metrics at P14, lungs were harvested for detection. (B) Representative images of H&E-stained lungs. The left panel shows low-magnification (scale bar = 100 μm) images, and the right panel shows higher-magnification (scale bar = 20 μm) images. (C,D) (Continued )

Article Snippet: Hyperoxia induces ECs loss and upregulates the expression of endothelial FGFR1 and the classic FGFR1 signaling pathways in ECs To investigate the cellular and molecular changes resulting from neonatal lung injury induced by hyperoxia, scRNA-seq was performed on a 10X genomics platform to generate scRNA-seq profiles of WT mice reared in normoxia or hyperoxia (Figure 2A).

Techniques: Staining

Journal: Frontiers in pharmacology

Article Title: Deficiency of endothelial FGFR1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice.

doi: 10.3389/fphar.2022.1039103

Figure Lengend Snippet: FIGURE 2 scRNA-seq analysis of lungs from normoxia- and hyperoxia-reared mice. (A) Approach to generate a single-cell atlas. (B) UMAP plot of all scRNA-seq data, showing a total of 19 distinct cell types corresponding to 5 major cell groups. Cell populations are colored as indicated by the legend. (C) Heatmap of the top 5 most differentially expressed genes across 5 major cell types. The intensity of expression is indicated as specified by the color legend. (D) Feature plots showing the expression of principal identifiers of epithelial cells, endothelial cells, stromal cells, myeloid cells, lymphocytes (B cells) and lymphocyte (T cells) populations. (E) Cellular compositions are colored as indicated by the legend in normal and hyperoxia- (Continued )

Article Snippet: Hyperoxia induces ECs loss and upregulates the expression of endothelial FGFR1 and the classic FGFR1 signaling pathways in ECs To investigate the cellular and molecular changes resulting from neonatal lung injury induced by hyperoxia, scRNA-seq was performed on a 10X genomics platform to generate scRNA-seq profiles of WT mice reared in normoxia or hyperoxia (Figure 2A).

Techniques: Expressing

Journal: Frontiers in pharmacology

Article Title: Deficiency of endothelial FGFR1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice.

doi: 10.3389/fphar.2022.1039103

Figure Lengend Snippet: FIGURE 3 RNA-seq analysis of differentially expressed genes (DEGs) and hyperoxia-impacted signaling pathways in ECs from normal and hyperoxia- impaired lungs. (A) Volcano plot showing upregulated and downregulated transcript levels of DEGs, p-value < 0.05, |log2FoldChange|≥1. (B) Heatmap of the top 50 upregulated DEGs and top 50 downregulated DEGs. (C) Hyperoxia-impacted signaling pathways in ECs as identified by GO enrichment analysis of biological processes. All terms shown are significantly enriched (p-value < 0.05). (D) Hyperoxia-impacted signaling pathways in ECs as identified by KEGG pathway enrichment analysis. All terms shown are significantly enriched (p-value < 0.05). (E) Upregulated downstream pathways of activated FGFR1 as revealed by Gene Set Enrichment Analysis, all terms shown are significantly enriched (p-value < 0.05). (F) qPCR of Fgfr1 expression in ECs of normoxic or hyperoxic lungs. n = 7 per group. Data are shown as means ± SEMs. **p < 0.01. (G) Western blot examining the expression of FGFR1 in ECs of normoxic or hyperoxic lung, Gapdh as negative control.

Article Snippet: Hyperoxia induces ECs loss and upregulates the expression of endothelial FGFR1 and the classic FGFR1 signaling pathways in ECs To investigate the cellular and molecular changes resulting from neonatal lung injury induced by hyperoxia, scRNA-seq was performed on a 10X genomics platform to generate scRNA-seq profiles of WT mice reared in normoxia or hyperoxia (Figure 2A).

Techniques: RNA Sequencing, Protein-Protein interactions, Expressing, Western Blot, Negative Control

Journal: Frontiers in pharmacology

Article Title: Deficiency of endothelial FGFR1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice.

doi: 10.3389/fphar.2022.1039103

Figure Lengend Snippet: FIGURE 4 Deletion of endothelial Fgfr1 improved alveolar development and respiratory metrics and angiogenesis in mice upon hyperoxia. (A) Schematic representation of EC-specific inducible deletion of Fgfr1 in neonatal mice. (B) Representative images of H&E-stained lungs from Fgfr1 +/+ and Fgfr1iΔEC/iΔEC mice reared in room air or hyperoxia. The top panel shows low-magnification (scale bar = 100 μm) images, and the bottom panel shows higher-magnification (scale bar = 20 μm) images. (C,D) Quantification of MLI (C) and RAC (D) based on the data in (B). Data are shown as (Continued )

Article Snippet: Hyperoxia induces ECs loss and upregulates the expression of endothelial FGFR1 and the classic FGFR1 signaling pathways in ECs To investigate the cellular and molecular changes resulting from neonatal lung injury induced by hyperoxia, scRNA-seq was performed on a 10X genomics platform to generate scRNA-seq profiles of WT mice reared in normoxia or hyperoxia (Figure 2A).

Techniques: Staining

Journal: Frontiers in pharmacology

Article Title: Deficiency of endothelial FGFR1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice.

doi: 10.3389/fphar.2022.1039103

Figure Lengend Snippet: FIGURE 5 Hyperoxia induced upregulation of FGFR1 mainly in aCap cells rather than in gCap cells. (A) A total of 5 clusters of ECs were identified. Cell populations are colored as indicated by the legend. (B) Feature plots showing the expression of principal identifiers of general capillary endothelial cells (gCap), aerocyte capillary endothelial cells (aCap), arterial endothelial cells (Artery), venous endothelial cells (Vein) and lymphatic endothelial cells (Lymph). (C) Dotplot depicting the top 5 most differentially expressed genes across endothelial clusters. The intensity of expression is (Continued )

Article Snippet: Hyperoxia induces ECs loss and upregulates the expression of endothelial FGFR1 and the classic FGFR1 signaling pathways in ECs To investigate the cellular and molecular changes resulting from neonatal lung injury induced by hyperoxia, scRNA-seq was performed on a 10X genomics platform to generate scRNA-seq profiles of WT mice reared in normoxia or hyperoxia (Figure 2A).

Techniques: Expressing

Journal: Frontiers in pharmacology

Article Title: Deficiency of endothelial FGFR1 alleviates hyperoxia-induced bronchopulmonary dysplasia in neonatal mice.

doi: 10.3389/fphar.2022.1039103

Figure Lengend Snippet: FIGURE 6 Inhibition of endothelial Fgfr1 improved alveolar development and respiratory metrics in neonatal mice in hyperoxia. (A) Representative images of H&E-stained lungs from vehicle or FGFR1 inhibitor treated mice. The top panel shows low-magnification (scale bar = 100 μm) images, and the bottom panel shows higher-magnification (scale bar = 20 μm) images. (B,C) Quantification of MLI (B) and RAC (C) based on the data in (A). Data are shown as means ± SEMs. n = 6 per group. **p < 0.01, ***p < 0.001, ****p < 0.0001. (D–G) Results of respiratory metrics measurement. Data are shown as means ± SEMs. n = 10 per group. *p < 0.05, **p < 0.01, ****p < 0.0001.

Article Snippet: Hyperoxia induces ECs loss and upregulates the expression of endothelial FGFR1 and the classic FGFR1 signaling pathways in ECs To investigate the cellular and molecular changes resulting from neonatal lung injury induced by hyperoxia, scRNA-seq was performed on a 10X genomics platform to generate scRNA-seq profiles of WT mice reared in normoxia or hyperoxia (Figure 2A).

Techniques: Inhibition, Staining