Susceptibility to Crohn's disease (CD), a complex inflammatory disease involving the small intestine, is controlled by up to 32 loci1. One CD risk allele is in ATG16L1, a gene homologous to the essential yeast autophagy gene ATG162. It is not known how Atg16L1 or autophagy contributes to intestinal biology or CD pathogenesis. To address these questions we generated and characterized mice that are hypomorphic for Atg16L1 protein expression, and validated conclusions based on studies in these mice by analyzing intestinal tissues that we collected from CD patients carrying the CD risk allele of ATG16L1. We show that Atg16L1 is a bona fide autophagy protein. Within the ileal epithelium, both Atg16L1 and a second essential autophagy protein Atg5 are selectively important for the biology of the Paneth cell, a specialized epithelial cell which functions in part by secretion of granule contents containing antimicrobial peptides and other proteins that alter the intestinal environment3. Atg16L1 and Atg5-deficient Paneth cells exhibited striking abnormalities in the granule exocytosis pathway. In addition, transcriptional analysis revealed an unexpected gain of function specific to Atg16L1-deficient Paneth cells including increased expression of genes involved in PPAR signaling and lipid metabolism, acute phase reactants, as well as two adipocytokines, leptin and adiponectin, known to directly influence intestinal injury responses. Importantly, CD patients homozygous for the ATG16L1 CD risk allele displayed Paneth cell granule abnormalities similar to those observed in autophagy protein-deficient mice and expressed increased levels of leptin protein. Thus, Atg16L1, and likely the process of autophagy, play their role within the intestinal epithelium of mice and CD patients by selective effects on the cell biology and specialized regulatory properties of Paneth cells.
Pancreatic cancer is the most lethal common solid malignancy. Systemic therapies are often ineffective, and predictive biomarkers to guide treatment are urgently needed. We generated a pancreatic cancer patient-derived organoid (PDO) library that recapitulates the mutational spectrum and transcriptional subtypes of primary pancreatic cancer. New driver oncogenes were nominated and transcriptomic analyses revealed unique clusters. PDOs exhibited heterogeneous responses to standard-of-care chemotherapeutics and investigational agents. In a case study manner, we found that PDO therapeutic profiles paralleled patient outcomes and that PDOs enabled longitudinal assessment of chemosensitivity and evaluation of synchronous metastases. We derived organoid-based gene expression signatures of chemosensitivity that predicted improved responses for many patients to chemotherapy in both the adjuvant and advanced disease settings. Finally, we nominated alternative treatment strategies for chemorefractory PDOs using targeted agent therapeutic profiling. We propose that combined molecular and therapeutic profiling of PDOs may predict clinical response and enable prospective therapeutic selection. New approaches to prioritize treatment strategies are urgently needed to improve survival and quality of life for patients with pancreatic cancer. Combined genomic, transcriptomic, and therapeutic profiling of PDOs can identify molecular and functional subtypes of pancreatic cancer, predict therapeutic responses, and facilitate precision medicine for patients with pancreatic cancer. .
Background-Abnormal host-microbe interactions are implicated in the pathogenesis of inflammatory bowel diseases. Previous 16S rRNA sequence analysis of intestinal tissues demonstrated that a subset of Crohn's disease (CD) and ulcerative colitis (UC) samples exhibited altered intestinal associated microbial compositions characterized by depletion of Bacteroidetes and Firmicutes (particularly Clostridium taxa). We hypothesize that NOD2 and ATG16L1 risk
In order to assess potential associations between autism spectrum disorder (ASD) phenotype, functional GI disorders and fecal microbiota, we recruited simplex families, which had only a single ASD proband and neurotypical (NT) siblings, through the Simons Simplex Community at the Interactive Autism Network (SSC@IAN). Fecal samples and metadata related to functional GI disorders and diet were collected from ASD probands and NT siblings of ASD probands (age 7–14). Functional gastrointestinal disorders (FGID) were assessed using the parent-completed ROME III questionnaire for pediatric FGIDs, and problem behaviors were assessed using the Child Behavior Check List (CBCL). Targeted quantitative polymerase chain reaction (qPCR) assays were conducted on selected taxa implicated in ASD, including Sutterella spp., Bacteroidetes spp. and Prevotella spp. Illumina sequencing of the V1V2 and the V1V3 regions of the bacterial 16S rRNA genes from fecal DNA was performed to an average depth of 208,000 and 107,000 high-quality reads respectively. Twenty-five of 59 ASD children and 13 of 44 NT siblings met ROME III criteria for at least one FGID. Functional constipation was more prevalent in ASD (17 of 59) compared to NT siblings (6 of 44, P = 0.035). The mean CBCL scores in NT siblings with FGID, ASD children with FGID and ASD without FGID were comparably higher (58–62 vs. 44, P < 0.0001) when compared to NT children without FGID. There was no significant difference in macronutrient intake between ASD and NT siblings. There was no significant difference in ASD severity scores between ASD children with and without FGID. No significant difference in diversity or overall microbial composition was detected between ASD children with NT siblings. Exploratory analysis of the 16S rRNA sequencing data, however, identified several low abundance taxa binned at the genus level that were associated with ASD and/or first order ASD*FGID interactions (FDR <0.1).
We tested the hypothesis that Crohn’s disease (CD)-related genetic polymorphisms involved in host innate immunity are associated with shifts in human ileum–associated microbial composition in a cross-sectional analysis of human ileal samples. Sanger sequencing of the bacterial 16S ribosomal RNA (rRNA) gene and 454 sequencing of 16S rRNA gene hypervariable regions (V1–V3 and V3–V5), were conducted on macroscopically disease-unaffected ileal biopsies collected from 52 ileal CD, 58 ulcerative colitis and 60 control patients without inflammatory bowel diseases (IBD) undergoing initial surgical resection. These subjects also were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R) and the ATG16L1 risk allele (T300A). The samples were linked to clinical metadata, including body mass index, smoking status and Clostridia difficile infection. The sequences were classified into seven phyla/subphyla categories using the Naïve Bayesian Classifier of the Ribosome Database Project. Centered log ratio transformation of six predominant categories was included as the dependent variable in the permutation based MANCOVA for the overall composition with stepwise variable selection. Polymerase chain reaction (PCR) assays were conducted to measure the relative frequencies of the Clostridium coccoides – Eubacterium rectales group and the Faecalibacterium prausnitzii spp. Empiric logit transformations of the relative frequencies of these two microbial groups were included in permutation-based ANCOVA. Regardless of sequencing method, IBD phenotype, Clostridia difficile and NOD2 genotype were selected as associated (FDR ≤0.05) with shifts in overall microbial composition. IBD phenotype and NOD2 genotype were also selected as associated with shifts in the relative frequency of the C. coccoides – E. rectales group. IBD phenotype, smoking and IBD medications were selected as associated with shifts in the relative frequency of F. prausnitzii spp. These results indicate that the effects of genetic and environmental factors on IBD are mediated at least in part by the enteric microbiota.
The intestine and other tissues are able to synthesize retinyl esters in an acyl-CoA-dependent manner involving an acylCoA:retinol acyltransferase (ARAT). However, the molecular identity of this ARAT has not been established. Recent studies of lecithin:retinol acyltransferase (LRAT)-deficient mice indicate that LRAT is responsible for the preponderance of retinyl ester synthesis in the body, aside from in the intestine and adipose tissue. Our present studies, employing a number of mutant mouse models, identify diacylglycerol acyltransferase 1 (DGAT1) as an important intestinal ARAT in vivo. The contribution that DGAT1 makes to intestinal retinyl ester synthesis becomes greater when a large pharmacologic dose of retinol is administered by gavage to mice. Moreover, when large retinol doses are administered another intestinal enzyme(s) with ARAT activity becomes apparent. Surprisingly, although DGAT1 is expressed in adipose tissue, DGAT1 does not catalyze retinyl ester synthesis in adipose tissue in vivo. Our data also establish that cellular retinol-binding protein, type II (CRBPII), which is expressed solely in the adult intestine, in vivo channels retinol to LRAT for retinyl ester synthesis. Contrary to what has been proposed in the literature based on in vitro studies, CRBPII does not directly prevent retinol from being acted upon by DGAT1 or other intestinal ARATs in vivo.
Culture-independent microbiological technologies that interrogate complex microbial populations without prior axenic culture, coupled with high-throughput DNA sequencing, have revolutionized the scale, speed, and economics of microbial ecological studies. Their application to the medical realm has lead to a highly productive merger of clinical, experimental, and environmental microbiology. The functional roles played by members of the human microbiota are being actively explored through experimental manipulation of animal model systems and studies of human populations. In concert, these studies have appreciably expanded our understanding of the composition and dynamics of human-associated microbial communities (microbiota). Of note, several human diseases have been linked to alterations in the composition of resident microbial communities, so-called dysbiosis [1]. However, how changes in microbial communities contribute to disease etiology remains poorly defined. Correlation of microbial composition represents integration of only two datasets (phenotype and microbial composition). This article explores strategies for merging the human microbiome data with multiple additional datasets (e.g. host single nucleotide polymorphisms [SNP] and host gene expression) and for integrating patient-based data with results from experimental animal models to gain deeper understanding of how host-microbe interactions impact disease.
Pancreatic cancer organoids can be successfully and rapidly created by means of EUS-FNB sampling using a 22-gauge needle at the time of initial diagnosis. Successful organoid generation is essential for precision medicine in patients with pancreatic cancer in whom most are not surgically resectable. (Clinical trial registration number: NCT03140592.).
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