Akkermansia muciniphila has received great attention because of its beneficial roles in gut health by regulating gut immunity, promoting intestinal epithelial development, and improving barrier ...integrity. However, A. muciniphila-derived functional molecules regulating gut health are not well understood. Microbiome-secreted proteins act as key arbitrators of host-microbiome crosstalk through interactions with host cells in the gut and are important for understanding host-microbiome relationships. Herein, we report the biological function of Amuc_1409, a previously uncharacterised A. muciniphila-secreted protein. Amuc_1409 increased intestinal stem cell (ISC) proliferation and regeneration in ex vivo intestinal organoids and in vivo models of radiation- or chemotherapeutic drug-induced intestinal injury and natural aging with male mice. Mechanistically, Amuc_1409 promoted E-cadherin/β-catenin complex dissociation via interaction with E-cadherin, resulting in the activation of Wnt/β-catenin signaling. Our results demonstrate that Amuc_1409 plays a crucial role in intestinal homeostasis by regulating ISC activity in an E-cadherin-dependent manner and is a promising biomolecule for improving and maintaining gut health.
Abstract
Background
Inflammatory bowel disease (IBD) is a chronic and idiopathic inflammatory disorder of the gastrointestinal tract and comprises ulcerative colitis (UC) and Crohn’s disease (CD). ...Crohn’s disease can affect any part of the gastrointestinal tract, but mainly the terminal ileum and colon. In the present study, we aimed to characterize terminal-ileal CD (ICD) and colonic CD (CCD) at the molecular level, which might enable a more optimized approach for the clinical care and scientific research of CD.
Methods
We analyzed differentially expressed genes in samples from 23 treatment-naïve paediatric patients with CD and 25 non-IBD controls, and compared the data with previously published RNA-Seq data using multi-statistical tests and confidence intervals. We implemented functional profiling and proposed statistical methods for feature selection using a logistic regression model to identify genes that are highly associated in ICD or CCD. We also validated our final candidate genes in independent paediatric and adult cohorts.
Results
We identified 550 genes specifically expressed in patients with CD compared with those in healthy controls (p < 0.05). Among these DEGs, 240 from patients with CCD were mainly involved in mitochondrial dysfunction, whereas 310 from patients with ICD were enriched in the ileum functions such as digestion, absorption, and metabolism. To choose the most effective gene set, we selected the most powerful genes (
p
-value ≤ 0.05, accuracy ≥ 0.8, and AUC ≥ 0.8) using logistic regression. Consequently, 33 genes were identified as useful for discriminating CD location; the accuracy and AUC were 0.86 and 0.83, respectively. We then validated the 33 genes with data from another independent paediatric cohort (accuracy = 0.93, AUC = 0.92) and adult cohort (accuracy = 0.88, AUC = 0.72).
Conclusions
In summary, we identified DEGs that are specifically expressed in CCD and ICD compared with those in healthy controls and patients with UC. Based on the feature selection analysis, 33 genes were identified as useful for discriminating CCD and ICD with high accuracy and AUC, for not only paediatric patients but also independent cohorts. We propose that our approach and the final gene set are useful for the molecular classification of patients with CD, and it could be beneficial in treatments based on disease location.
For current and future applications of human intestinal organoids (hIOs) to various aspects of in vivo research and their potential clinical use, an efficient noninvasive system is needed to directly ...visualize the stage of intestinal differentiation and graft-host interactions and for further safety monitoring and efficacy. Here, we describe a detailed method for monitoring and histologically identifying implanted hIO-expressing eGFP and mCherry fluorescence under the kidney capsule of immunodeficient mice with fluorescence imaging (FLI). We then describe the orthotropic transplantation method of hIOs and methods to confirm successful engraftment in the small intestines of immunodeficient mice. These methods provide an approach for tracking the location of intestinal cells in hIOs in vivo and ex vivo using a fluorescent reporter system from the beginning of engraftment to various subsequent experiments.
Avian pathogenic Escherichia coli (APEC) causes extensive mortality in poultry flocks, leading to extensive economic losses. The aim of this study was to investigate the phenotypic and genotypic ...characteristics and antimicrobial resistance of recent APEC isolates. Of the 79 APEC isolates, the most predominant serogroup was O78 (16 isolates, 20.3%), followed by O2 (7 isolates, 8.9%) and O53 (7 isolates, 8.9%). Thirty-seven (46.8%) and six (7.6%) of the isolates belonged to phylogenetic groups D and B2, respectively, and presented as virulent extraintestinal E. coli. Among 5 analyzed virulence genes, the highest frequency was observed in hlyF (74 isolates, 93.7%), followed by iutA (72 isolates, 91.9%) gene. The distribution of the iss gene was significantly different between groups A/B1 and B2/D (P < 0.05). All group B2 isolates carried all 5 virulence genes. APEC isolates showed high resistance to ampicillin (83.5%), nalidixic acid (65.8%), tetracycline (64.6%), cephalothin (46.8%), and ciprofloxacin (46.8%). The β-lactamases–encoding genes blaTEM-1 (23 isolates, 29.1%), blaCTX-M-1 (4 isolates, 5.1%), and blaCTX-M-15 (3 isolates, 3.8%); the aminoglycoside-modifying enzyme gene aac(3)-II (4 isolates, 5.1%); and the plasmid-mediated quinolone genes qnrA (10 isolates, 12.7%) and qnrS (2 isolates, 2.5%) were identified in APEC isolates. The tetA (37 isolates, 46.8%) and sul2 (20 isolates, 25.3%) were the most prevalent among tetracycline and sulfonamide resistant isolates, respectively. This study indicates that APEC isolates harbor a variety of virulence and resistance genes; such genes are often associated with plasmids that facilitate their transmission between bacteria and should be continuously monitored to track APEC transmission in poultry farms.
Innovative advances in stem cell research have resulted in the development of organoids, which are widely used as
models of human organ development and for disease. The long-term goals of scientists ...include the generation of high-quality organoids with properties like those of native organs, and to expand their use to a variety of applications such as drug discovery and organoid-based cell therapy. In particular, the combination of human induced pluripotent stem cell (iPSC)-derived organoids with the recently developed genome engineering, biotechnology serve as an attractive platform in precision medicine. This review briefly summarizes the generation of organoids derived mostly from iPSCs without ethical issues, and describes the applications and technological advances of organoids under their differentiation and culture conditions. We also discuss the approaches to improve the organoid models, and how organoids can recapitulate mature organ systems of the human body for regenerative medicine. Finally, the future perspectives and remaining challenges in the field have been discussed to provide a better understanding of the potential applications of organoids.
Reprogramming with episomal vectors is an easy, safe, and cost-effective method to generate exogenous DNA-free (exogene-free) induced pluripotent stem cells (iPSCs). However, the genomic integration ...of exogenes is observed occasionally. Additionally, the removal of episomal DNA takes more than 70 days in established iPSCs. Here, we inserted the cytosine deaminase (CD) gene from yeast into episomal vectors and used them to reprogram human fibroblasts into iPSCs. These new episomal vectors (CD episomal vectors) were eliminated from the generated iPSCs as early as seven days after 5-fluorocytosine (5-FC) treatment. We also found that cells with the integration of the CD gene perished within two days of 5-FC treatment. In addition, we generated exogene-free induced neural stem cells after one passage by direct reprogramming with CD episomal vectors combined with 5-FC treatment. Conclusively, our novel method allows the rapid and easy isolation of exogene-free reprogrammed cells and can be applied to disease modeling and clinical applications.
Genomic changes frequently occur in cancer cells during tumorigenesis from normal cells. Using the Illumina Human NS-12 single-nucleotide polymorphism (SNP) chip to screen for gene copy number ...changes in primary hepatocellular carcinomas (HCCs), we initially detected amplification of 35 genes from four genomic regions (1q21-41, 6p21.2-24.1, 7p13 and 8q13-23). By integrated screening of these genes for both DNA copy number and gene expression in HCC and colorectal cancer, we selected CENPF (centromere protein F/mitosin), GMNN (geminin, DNA replication inhibitor), CDK13 (cyclin-dependent kinase 13), and FAM82B (family with sequence similarity 82, member B) as common cancer genes. Each gene exhibited an amplification frequency of ~30% (range, 20-50%) in primary HCC (n = 57) and colorectal cancer (n = 12), as well as in a panel of human cancer cell lines (n = 70). Clonogenic and invasion assays of NIH3T3 cells transfected with each of the four amplified genes showed that CENPF, GMNN, and CDK13 were highly oncogenic whereas FAM82B was not. Interestingly, the oncogenic activity of these genes (excluding FAM82B) was highly correlated with gene-copy numbers in tumor samples (correlation coefficient, r>0.423), indicating that amplifications of CENPF, GMNN, and CDK13 genes are tightly linked and coincident in tumors. Furthermore, we confirmed that CDK13 gene copy number was significantly associated with clinical onset age in patients with HCC (P = 0.0037). Taken together, our results suggest that coincidently amplified CDK13, GMNN, and CENPF genes can play a role as common cancer-driver genes in human cancers.
To exert their beneficial effects, it is essential for the commensal bacteria of probiotic supplements to be sufficiently protected as they pass through the low pH environment of the stomach, and ...effectively colonize the intestinal epithelium downstream. Here, we investigated the effect of a multilayer coating containing red ginseng dietary fiber, on the acid tolerance, and the adhesion and proliferation capacities of three
Lactobacillus
strains (
Limosilactobacillus reuteri
KGC1901,
Lacticaseibacillus casei
KGC1201,
Limosilactobacillus fermentum
KGC1601) isolated from
Panax ginseng
, using HT-29 cells, mucin-coated plates, and human pluripotent stem cell-derived intestinal epithelial cells as in vitro models of human gut physiology. We observed that the multilayer-coated strains displayed improved survival rates after passage through gastric juice, as well as high adhesion and proliferation capacities within the various gut epithelial systems tested, compared to their uncoated counterparts. Our findings demonstrated that the multilayer coat effectively protected commensal microbiota and led to improved adhesion and colonization of intestinal epithelial cells, and consequently to higher probiotic efficacy.
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