Supplementary Figure 2 from Identification of Actively Translated mRNA Transcripts in a Rat Model... more Supplementary Figure 2 from Identification of Actively Translated mRNA Transcripts in a Rat Model of Early-Stage Colon Carcinogenesis
Supplementary Figure 6 from Identification of Actively Translated mRNA Transcripts in a Rat Model... more Supplementary Figure 6 from Identification of Actively Translated mRNA Transcripts in a Rat Model of Early-Stage Colon Carcinogenesis
ScopeThis study investigates the mechanism of action and functional effects of coffee extracts in... more ScopeThis study investigates the mechanism of action and functional effects of coffee extracts in colonic cells, on intestinal stem cell growth, and inhibition of dextran sodium sulfate (DSS)‐induced intestinal barrier damage in mice.Methods and ResultsAqueous coffee extracts induced Ah receptor (AhR) ‐responsive CYP1A1, CYP1B1, and UGT1A1 gene expression in colon‐derived Caco2 and YAMC cells. Tissue‐specific AhR knockout (AhRf/f x Lgr5‐GFP‐CreERT2 x Villin‐Cre), wild‐type (Lgr5‐CreERT2 x Villin‐Cre) mice are sources of stem cell enriched organoids and both coffee extracts and norharman, an AhR‐active component of these extracts inhibited stem cell growth. Coffee extracts also inhibit DSS‐induced damage to intestinal barrier function and DSS‐induced mucosal inflammatory genes such as IL‐6 and TGF‐β1 in wild‐type (AhR+/+) but not AhR–/– mice. In contrast, coffee does not exhibit protective effects in intestinal‐specific AhR knockout mice. Coffee extracts also enhanced overall formation of AhR‐active microbial metabolites.ConclusionsIn colon‐derived cells and in the mouse intestine, coffee induced several AhR‐dependent responses including gene expression, inhibition of intestinal stem cell‐enriched organoid growth, and inhibition of DSS‐induced intestinal barrier damage. We conclude that the anti‐inflammatory effects of coffee in the intestine are due, in part, to activation of AhR signaling.
Intestinal organoids (IO), known as “mini-guts”, derived from intestinal crypts, are self-organiz... more Intestinal organoids (IO), known as “mini-guts”, derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO model for poultry research is lacking. Therefore, the objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3β inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.
Supplementary Table 2 from Noninvasive Detection of Candidate Molecular Biomarkers in Subjects wi... more Supplementary Table 2 from Noninvasive Detection of Candidate Molecular Biomarkers in Subjects with a History of Insulin Resistance and Colorectal Adenomas
Supplementary Figure 2 from Identification of Actively Translated mRNA Transcripts in a Rat Model... more Supplementary Figure 2 from Identification of Actively Translated mRNA Transcripts in a Rat Model of Early-Stage Colon Carcinogenesis
Supplementary Figure 6 from Identification of Actively Translated mRNA Transcripts in a Rat Model... more Supplementary Figure 6 from Identification of Actively Translated mRNA Transcripts in a Rat Model of Early-Stage Colon Carcinogenesis
ScopeThis study investigates the mechanism of action and functional effects of coffee extracts in... more ScopeThis study investigates the mechanism of action and functional effects of coffee extracts in colonic cells, on intestinal stem cell growth, and inhibition of dextran sodium sulfate (DSS)‐induced intestinal barrier damage in mice.Methods and ResultsAqueous coffee extracts induced Ah receptor (AhR) ‐responsive CYP1A1, CYP1B1, and UGT1A1 gene expression in colon‐derived Caco2 and YAMC cells. Tissue‐specific AhR knockout (AhRf/f x Lgr5‐GFP‐CreERT2 x Villin‐Cre), wild‐type (Lgr5‐CreERT2 x Villin‐Cre) mice are sources of stem cell enriched organoids and both coffee extracts and norharman, an AhR‐active component of these extracts inhibited stem cell growth. Coffee extracts also inhibit DSS‐induced damage to intestinal barrier function and DSS‐induced mucosal inflammatory genes such as IL‐6 and TGF‐β1 in wild‐type (AhR+/+) but not AhR–/– mice. In contrast, coffee does not exhibit protective effects in intestinal‐specific AhR knockout mice. Coffee extracts also enhanced overall formation of AhR‐active microbial metabolites.ConclusionsIn colon‐derived cells and in the mouse intestine, coffee induced several AhR‐dependent responses including gene expression, inhibition of intestinal stem cell‐enriched organoid growth, and inhibition of DSS‐induced intestinal barrier damage. We conclude that the anti‐inflammatory effects of coffee in the intestine are due, in part, to activation of AhR signaling.
Intestinal organoids (IO), known as “mini-guts”, derived from intestinal crypts, are self-organiz... more Intestinal organoids (IO), known as “mini-guts”, derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO model for poultry research is lacking. Therefore, the objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3β inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.
Supplementary Table 2 from Noninvasive Detection of Candidate Molecular Biomarkers in Subjects wi... more Supplementary Table 2 from Noninvasive Detection of Candidate Molecular Biomarkers in Subjects with a History of Insulin Resistance and Colorectal Adenomas
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