NAD+ precursors and bile acid sequestration treat preclinical refractory environmental enteric dysfunction.

Environmental enteric dysfunction (EED) is a diffuse small bowel disorder associated with poor growth, inadequate responses to oral vaccines, and nutrient malabsorption in millions of children worldwide. We identify loss of the small intestinal Paneth and goblet cells that are critical for innate immunity, reduced villous height, increased bile acids, and dysregulated nicotinamide adenine dinucleotide (NAD+) synthesis signaling as potential mechanisms underlying EED and which also correlated with diminished length-for-age z score. Isocaloric low-protein diet (LPD) consumption in mice recapitulated EED histopathology and transcriptomic changes in a microbiota-independent manner, as well as increases in serum and fecal bile acids. Children with refractory EED harbor single-nucleotide polymorphisms in key enzymes involved in NAD+ synthesis. In mice, deletion of Nampt, the gene encoding the rate-limiting enzyme in the NAD+ salvage pathway, from intestinal epithelium also reduced Paneth cell function, a deficiency that was further aggravated by LPD. Separate supplementation with NAD+ precursors or bile acid sequestrant partially restored LPD-associated Paneth cell defects and, when combined, fully restored all histopathology defects in LPD-fed mice. Therapeutic regimens that increase protein and NAD+ contents while reducing excessive bile acids may benefit children with refractory EED.

Distinct gene expression signatures comparing latent tuberculosis infection with different routes of Bacillus Calmette-Guérin vaccination.

Tuberculosis remains an international health threat partly because of limited protection from pulmonary tuberculosis provided by standard intradermal vaccination with Bacillus of Calmette and Guérin (BCG); this may reflect the inability of intradermal vaccination to optimally induce pulmonary immunity. In contrast, respiratory Mycobacterium tuberculosis infection usually results in the immune-mediated bacillary containment of latent tuberculosis infection (LTBI). Here we present RNA-Seq-based assessments of systemic and pulmonary immune cells from LTBI participants and recipients of intradermal and oral BCG. LTBI individuals uniquely display ongoing immune activation and robust CD4 T cell recall responses in blood and lung. Intradermal BCG is associated with robust systemic immunity but only limited pulmonary immunity. Conversely, oral BCG induces limited systemic immunity but distinct pulmonary responses including enhanced inflammasome activation potentially associated with mucosal-associated invariant T cells. Further, IL-9 is identified as a component of systemic immunity in LTBI and intradermal BCG, and pulmonary immunity following oral BCG.

Serum Proteomic Analysis of Peripartum Cardiomyopathy Reveals Distinctive Dysregulation of Inflammatory and Cholesterol Metabolism Pathways.

BACKGROUND: The pathophysiology of peripartum cardiomyopathy (PPCM) and its distinctive biological features remain incompletely understood. High-throughput serum proteomic profiling, a powerful tool to gain insights into the pathophysiology of diseases at a systems biology level, has never been used to investigate PPCM relative to nonischemic cardiomyopathy. OBJECTIVES: The aim of this study was to characterize the pathophysiology of PPCM through serum proteomic analysis. METHODS: Aptamer-based proteomic analysis (SomaScan 7K) was performed on serum samples from women with PPCM (n = 67), women with nonischemic nonperipartum cardiomyopathy (NPCM) (n = 31), and age-matched healthy peripartum and nonperipartum women (n = 10 each). Serum samples were obtained from the IPAC (Investigation of Pregnancy-Associated Cardiomyopathy) and IMAC2 (Intervention in Myocarditis and Acute Cardiomyopathy) studies. RESULTS: Principal component analysis revealed unique clustering of each patient group (P for difference <0.001). Biological pathway analyses of differentially measured proteins in PPCM relative to NPCM, before and after normalization to pertinent healthy controls, highlighted specific dysregulation of inflammatory pathways in PPCM, including the upregulation of the cholesterol metabolism-related anti-inflammatory pathway liver-X receptor/retinoid-X receptor (LXR/RXR) (P < 0.01, Z-score 1.9-2.1). Cardiac recovery by 12 months in PPCM was associated with the downregulation of pro-inflammatory pathways and the upregulation of LXR/RXR, and an additional RXR-dependent pathway involved in the regulation of inflammation and metabolism, peroxisome proliferator-activated receptor α/RXRα signaling. CONCLUSIONS: Serum proteomic profiling of PPCM relative to NPCM and healthy controls indicated that PPCM is a distinct disease entity characterized by the unique dysregulation of inflammation-related pathways and cholesterol metabolism-related anti-inflammatory pathways. These findings provide insight into the pathophysiology of PPCM and point to novel potential therapeutic targets.

Embryonic vitamin D deficiency programs hematopoietic stem cells to induce type 2 diabetes.

Environmental factors may alter the fetal genome to cause metabolic diseases. It is unknown whether embryonic immune cell programming impacts the risk of type 2 diabetes in later life. We demonstrate that transplantation of fetal hematopoietic stem cells (HSCs) made vitamin D deficient in utero induce diabetes in vitamin D-sufficient mice. Vitamin D deficiency epigenetically suppresses Jarid2 expression and activates the Mef2/PGC1a pathway in HSCs, which persists in recipient bone marrow, resulting in adipose macrophage infiltration. These macrophages secrete miR106-5p, which promotes adipose insulin resistance by repressing PIK3 catalytic and regulatory subunits and down-regulating AKT signaling. Vitamin D-deficient monocytes from human cord blood have comparable Jarid2/Mef2/PGC1a expression changes and secrete miR-106b-5p, causing adipocyte insulin resistance. These findings suggest that vitamin D deficiency during development has epigenetic consequences impacting the systemic metabolic milieu.

Corrigendum: The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies.

[This corrects the article DOI: 10.3389/fimmu.2022.1093242.].

Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia.

Enterotoxigenic E. coli (ETEC) produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and commonly cause diarrhea in resource-poor regions. ETEC have been linked repeatedly to sequelae in children including enteropathy, malnutrition, and growth impairment. Although cellular actions of ETEC enterotoxins leading to diarrhea are well-established, their contributions to sequelae remain unclear. LT increases cellular cAMP to activate protein kinase A (PKA) that phosphorylates ion channels driving intestinal export of salt and water resulting in diarrhea. As PKA also modulates transcription of many genes, we interrogated transcriptional profiles of LT-treated intestinal epithelia. Here we show that LT significantly alters intestinal epithelial gene expression directing biogenesis of the brush border, the major site for nutrient absorption, suppresses transcription factors HNF4 and SMAD4 critical to enterocyte differentiation, and profoundly disrupts microvillus architecture and essential nutrient transport. In addition, ETEC-challenged neonatal mice exhibit substantial brush border derangement that is prevented by maternal vaccination with LT. Finally, mice repeatedly challenged with toxigenic ETEC exhibit impaired growth recapitulating the multiplicative impact of recurring ETEC infections in children. These findings highlight impacts of ETEC enterotoxins beyond acute diarrheal illness and may inform approaches to prevent major sequelae of these common infections including malnutrition that impact millions of children.

The Vacc-SeqQC project: Benchmarking RNA-Seq for clinical vaccine studies.

Introduction: Over the last decade, the field of systems vaccinology has emerged, in which high throughput transcriptomics and other omics assays are used to probe changes of the innate and adaptive immune system in response to vaccination. The goal of this study was to benchmark key technical and analytical parameters of RNA sequencing (RNA-seq) in the context of a multi-site, double-blind randomized vaccine clinical trial. Methods: We collected longitudinal peripheral blood mononuclear cell (PBMC) samples from 10 subjects before and after vaccination with a live attenuated Francisella tularensis vaccine and performed RNA-Seq at two different sites using aliquots from the same sample to generate two replicate datasets (5 time points for 50 samples each). We evaluated the impact of (i) filtering lowly-expressed genes, (ii) using external RNA controls, (iii) fold change and false discovery rate (FDR) filtering, (iv) read length, and (v) sequencing depth on differential expressed genes (DEGs) concordance between replicate datasets. Using synthetic mRNA spike-ins, we developed a method for empirically establishing minimal read-count thresholds for maintaining fold change accuracy on a per-experiment basis. We defined a reference PBMC transcriptome by pooling sequence data and established the impact of sequencing depth and gene filtering on transcriptome representation. Lastly, we modeled statistical power to detect DEGs for a range of sample sizes, effect sizes, and sequencing depths. Results and Discussion: Our results showed that (i) filtering lowly-expressed genes is recommended to improve fold-change accuracy and inter-site agreement, if possible guided by mRNA spike-ins (ii) read length did not have a major impact on DEG detection, (iii) applying fold-change cutoffs for DEG detection reduced inter-set agreement and should be used with caution, if at all, (iv) reduction in sequencing depth had a minimal impact on statistical power but reduced the identifiable fraction of the PBMC transcriptome, (v) after sample size, effect size (i.e. the magnitude of fold change) was the most important driver of statistical power to detect DEG. The results from this study provide RNA sequencing benchmarks and guidelines for planning future similar vaccine studies.

BRAF mutations may identify a clinically distinct subset of glioblastoma.

Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Prior studies examining the mutational landscape of GBM revealed recurrent alterations in genes that regulate the same growth control pathways. To this regard, ~ 40% of GBM harbor EGFR alterations, whereas BRAF variants are rare. Existing data suggests that gain-of-function mutations in these genes are mutually exclusive. This study was designed to explore the clinical, pathological, and molecular differences between EGFR- and BRAF-mutated GBM. We reviewed retrospective clinical data from 89 GBM patients referred for molecular testing between November 2012 and December 2015. Differences in tumor mutational profile, location, histology, and survival outcomes were compared in patients with EGFR- versus BRAF-mutated tumors, and microarray data from The Cancer Genome Atlas was used to assess differential gene expression between the groups. Individuals with BRAF-mutant tumors were typically younger and survived longer relative to those with EGFR-mutant tumors, even in the absence of targeted treatments. BRAF-mutant tumors lacked distinct histomorphology but exhibited unique localization in the brain, typically arising adjacent to the lateral ventricles. Compared to EGFR- and IDH1-mutant tumors, BRAF-mutant tumors showed increased expression of genes related to a trophoblast-like phenotype, specifically HLA-G and pregnancy specific glycoproteins, that have been implicated in invasion and immune evasion. Taken together, these observations suggest a distinct clinical presentation, brain location, and gene expression profile for BRAF-mutant tumors. Pending further study, this may prove useful in the stratification and management of GBM.

Western diet induces Paneth cell defects through microbiome alterations and farnesoid X receptor and type I interferon activation.

Intestinal Paneth cells modulate innate immunity and infection. In Crohn's disease, genetic mutations together with environmental triggers can disable Paneth cell function. Here, we find that a western diet (WD) similarly leads to Paneth cell dysfunction through mechanisms dependent on the microbiome and farnesoid X receptor (FXR) and type I interferon (IFN) signaling. Analysis of multiple human cohorts suggests that obesity is associated with Paneth cell dysfunction. In mouse models, consumption of a WD for as little as 4 weeks led to Paneth cell dysfunction. WD consumption in conjunction with Clostridium spp. increased the secondary bile acid deoxycholic acid levels in the ileum, which in turn inhibited Paneth cell function. The process required excess signaling of both FXR and IFN within intestinal epithelial cells. Our findings provide a mechanistic link between poor diet and inhibition of gut innate immunity and uncover an effect of FXR activation in gut inflammation.

Crohn's disease-associated ATG16L1 T300A genotype is associated with improved survival in gastric cancer.

BACKGROUND: A non-synonymous single nucleotide polymorphism of the ATG16L1 gene, T300A, is a major Crohn's disease (CD) susceptibility allele, and is known to be associated with increased apoptosis induction in the small intestinal crypt base in CD subjects and mouse models. We hypothesized that ATG16L1 T300A genotype also correlates with increased tumor apoptosis and therefore could lead to superior clinical outcome in cancer subjects. METHODS: T300A genotyping by Taqman assay was performed for gastric carcinoma subjects who underwent resection from two academic medical centers. Transcriptomic analysis was performed by RNA-seq on formalin-fixed paraffin-embedded cancerous tissue. Tumor apoptosis and autophagy were determined by cleaved caspase-3 and p62 immunohistochemistry, respectively. The subjects' genotypes were correlated with demographics, various histopathologic features, transcriptome, and clinical outcome. FINDINGS: Of the 220 genotyped subjects, 163 (74%) subjects carried the T300A allele(s), including 55 (25%) homozygous and 108 (49%) heterozygous subjects. The T300A/T300A subjects had superior overall survival than the other groups. Their tumors were associated with increased CD-like lymphoid aggregates and increased tumor apoptosis without concurrent increase in tumor mitosis or defective autophagy. Transcriptomic analysis showed upregulation of WNT/ß-catenin signaling and downregulation of PPAR, EGFR, and inflammatory chemokine pathways in tumors of T300A/T300A subjects. INTERPRETATION: Gastric carcinoma of subjects with the T300A/T300A genotype is associated with repressed EGFR and PPAR pathways, increased tumor apoptosis, and improved overall survival. Genotyping gastric cancer subjects may provide additional insight for clinical stratification.

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions for optimal therapeutic protection.

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes and CD8+ T cells for optimal clinical and virological benefit. Thus, potently neutralizing mAbs utilize Fc effector functions during therapy to mitigate lung infection and disease.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

BACKGROUND: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. METHODS: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. RESULTS: The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using 3 readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. CONCLUSIONS: Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

Proteomic Signatures of Heart Failure in Relation to Left Ventricular Ejection Fraction.

BACKGROUND: There is a growing recognition of the inherent limitations of the use of the left ventricular ejection fraction (LVEF) to accurately phenotype patients with heart failure (HF). OBJECTIVES: The authors sought to identify unique proteomic signatures for patients with HF with reduced ejection fraction (HFrEF), HF with a midrange LVEF (HFmrEF), and HF with preserved ejection fraction (HFpEF), as well as to identify molecular differences between patients with ischemic and nonischemic HF. METHODS: We used high-content aptamer-based proteomics technology (SOMAscan) to interrogate the blood proteome of age- and sex-matched patients with HF within different LVEF groups. RESULTS: Within the Washington University Heart Failure Registry, we identified age/sex-matched patients within 3 LVEF categories: HFrEF (LVEF <40%), HFmrEF (LVEF 40% to 50%), and HFpEF (LVEF >50%). We found that patients with HFrEF, HFmrEF, and HFpEF had unique variations in circulating proteins that reflected distinct biological pathophysiologies. Bioinformatics analysis revealed that there were biological themes that were unique to patients with HFrEF, HFpEF, or HFmrEF. Comparative analyses of patients with HFmrEF with improved LVEF and patients with HFmrEF with unchanged LVEF revealed marked differences between these 2 patient populations and indicated that patients with recovered LVEF are more similar to patients with HFpEF than to patients with HFrEF. Moreover, there were marked differences in the proteomic signatures of patients with ischemic and nonischemic HF. CONCLUSIONS: Viewed together, these findings suggest that it may be possible to use high-content multiplexed proteomics assays in combination with the clinical assessment of LVEF to more accurately identify clinical phenotypes of patients with HF.

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pretreatment protocols that enabled rapid and sensitive detection of < 102 viral genomes per reaction in contrived saliva controls. Moreover, our saliva pretreatment protocol enabled sensitive viral detection by conventional quantitative reverse transcription polymerase chain reaction (qRT-PCR) without RNA extraction. We validated the high performance of these assays on clinical samples and demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

Myeloid-specific Asxl2 deletion limits diet-induced obesity by regulating energy expenditure.

We previously established that global deletion of the enhancer of trithorax and polycomb (ETP) gene, Asxl2, prevents weight gain. Because proinflammatory macrophages recruited to adipose tissue are central to the metabolic complications of obesity, we explored the role of ASXL2 in myeloid lineage cells. Unexpectedly, mice without Asxl2 only in myeloid cells (Asxl2ΔLysM) were completely resistant to diet-induced weight gain and metabolically normal despite increased food intake, comparable activity, and equivalent fecal fat. Asxl2ΔLysM mice resisted HFD-induced adipose tissue macrophage infiltration and inflammatory cytokine gene expression. Energy expenditure and brown adipose tissue metabolism in Asxl2ΔLysM mice were protected from the suppressive effects of HFD, a phenomenon associated with relatively increased catecholamines likely due to their suppressed degradation by macrophages. White adipose tissue of HFD-fed Asxl2ΔLysM mice also exhibited none of the pathological remodeling extant in their control counterparts. Suppression of macrophage Asxl2 expression, via nanoparticle-based siRNA delivery, prevented HFD-induced obesity. Thus, ASXL2 controlled the response of macrophages to dietary factors to regulate metabolic homeostasis, suggesting modulation of the cells' inflammatory phenotype may impact obesity and its complications.

Human neutralizing antibodies against SARS-CoV-2 require intact Fc effector functions and monocytes for optimal therapeutic protection.

SARS-CoV-2 has caused the global COVID-19 pandemic. Although passively delivered neutralizing antibodies against SARS-CoV-2 show promise in clinical trials, their mechanism of action in vivo is incompletely understood. Here, we define correlates of protection of neutralizing human monoclonal antibodies (mAbs) in SARS-CoV-2-infected animals. Whereas Fc effector functions are dispensable when representative neutralizing mAbs are administered as prophylaxis, they are required for optimal protection as therapy. When given after infection, intact mAbs reduce SARS-CoV-2 burden and lung disease in mice and hamsters better than loss-of-function Fc variant mAbs. Fc engagement of neutralizing antibodies mitigates inflammation and improves respiratory mechanics, and transcriptional profiling suggests these phenotypes are associated with diminished innate immune signaling and preserved tissue repair. Immune cell depletions establish that neutralizing mAbs require monocytes for therapeutic efficacy. Thus, potently neutralizing mAbs require Fc effector functions for maximal therapeutic benefit during therapy to modulate protective immune responses and mitigate lung disease.

Effects of microbiota-directed foods in gnotobiotic animals and undernourished children.

To examine the contributions of impaired gut microbial community development to childhood undernutrition, we combined metabolomic and proteomic analyses of plasma samples with metagenomic analyses of fecal samples to characterize the biological state of Bangladeshi children with severe acute malnutrition (SAM) as they transitioned, after standard treatment, to moderate acute malnutrition (MAM) with persistent microbiota immaturity. Host and microbial effects of microbiota-directed complementary food (MDCF) prototypes targeting weaning-phase bacterial taxa underrepresented in SAM and MAM microbiota were characterized in gnotobiotic mice and gnotobiotic piglets colonized with age- and growth-discriminatory bacteria. A randomized, double-blind controlled feeding study identified a lead MDCF that changes the abundances of targeted bacteria and increases plasma biomarkers and mediators of growth, bone formation, neurodevelopment, and immune function in children with MAM.

Mechanisms by which sialylated milk oligosaccharides impact bone biology in a gnotobiotic mouse model of infant undernutrition.

Undernutrition in children is a pressing global health problem, manifested in part by impaired linear growth (stunting). Current nutritional interventions have been largely ineffective in overcoming stunting, emphasizing the need to obtain better understanding of its underlying causes. Treating Bangladeshi children with severe acute malnutrition with therapeutic foods reduced plasma levels of a biomarker of osteoclastic activity without affecting biomarkers of osteoblastic activity or improving their severe stunting. To characterize interactions among the gut microbiota, human milk oligosaccharides (HMOs), and osteoclast and osteoblast biology, young germ-free mice were colonized with cultured bacterial strains from a 6-mo-old stunted infant and fed a diet mimicking that consumed by the donor population. Adding purified bovine sialylated milk oligosaccharides (S-BMO) with structures similar to those in human milk to this diet increased femoral trabecular bone volume and cortical thickness, reduced osteoclasts and their bone marrow progenitors, and altered regulators of osteoclastogenesis and mediators of Th2 responses. Comparisons of germ-free and colonized mice revealed S-BMO-dependent and microbiota-dependent increases in cecal levels of succinate, increased numbers of small intestinal tuft cells, and evidence for activation of a succinate-induced tuft cell signaling pathway linked to Th2 immune responses. A prominent fucosylated HMO, 2'-fucosyllactose, failed to elicit these changes in bone biology, highlighting the structural specificity of the S-BMO effects. These results underscore the need to further characterize the balance between, and determinants of, osteoclastic and osteoblastic activity in stunted infants/children, and suggest that certain milk oligosaccharides may have therapeutic utility in this setting.

A multi-amplicon 16S rRNA sequencing and analysis method for improved taxonomic profiling of bacterial communities.

Metagenomic sequencing of bacterial samples has become the gold standard for profiling microbial populations, but 16S rRNA profiling remains widely used due to advantages in sample throughput, cost, and sensitivity even though the approach is hampered by primer bias and lack of specificity. We hypothesized that a hybrid approach, that combined targeted PCR amplification with high-throughput sequencing of multiple regions of the genome, would capture many of the advantages of both approaches. We developed a method that identifies and quantifies members of bacterial communities through simultaneous analysis of multiple variable regions of the bacterial 16S rRNA gene. The method combines high-throughput microfluidics for PCR amplification, short read DNA sequencing, and a custom algorithm named MVRSION (Multiple 16S Variable Region Species-Level IdentificatiON) for optimizing taxonomic assignment. MVRSION performance was compared to single variable region analyses (V3 or V4) of five synthetic mixtures of human gut bacterial strains using existing software (QIIME), and the results of community profiling by shotgun sequencing (COPRO-Seq) of fecal DNA samples collected from gnotobiotic mice colonized with a defined, phylogenetically diverse consortium of human gut bacterial strains. Positive predictive values for MVRSION ranged from 65%-91% versus 44%-61% for single region QIIME analyses (p < .01, p < .001), while the abundance estimate r2 for MVRSION compared to COPRO-Seq was 0.77 vs. 0.46 and 0.45 for V3-QIIME and V4-QIIME, respectively. MVRSION represents a generally applicable tool for taxonomic classification that is superior to single-region 16S rRNA methods, resource efficient, highly scalable for assessing the microbial composition of up to thousands of samples concurrently, with multiple applications ranging from whole community profiling to targeted tracking of organisms of interest in diverse habitats as a function of specified variables/perturbations.

Interaction between smoking and ATG16L1T300A triggers Paneth cell defects in Crohn's disease.

It is suggested that subtyping of complex inflammatory diseases can be based on genetic susceptibility and relevant environmental exposure (G+E). We propose that using matched cellular phenotypes in human subjects and corresponding preclinical models with the same G+E combinations is useful to this end. As an example, defective Paneth cells can subtype Crohn's disease (CD) subjects; Paneth cell defects have been linked to multiple CD susceptibility genes and are associated with poor outcome. We hypothesized that CD susceptibility genes interact with cigarette smoking, a major CD environmental risk factor, to trigger Paneth cell defects. We found that both CD subjects and mice with ATG16L1T300A (T300A; a prevalent CD susceptibility allele) developed Paneth cell defects triggered by tobacco smoke. Transcriptional analysis of full-thickness ileum and Paneth cell-enriched crypt base cells showed the T300A-smoking combination altered distinct pathways, including proapoptosis, metabolic dysregulation, and selective downregulation of the PPARγ pathway. Pharmacologic intervention by either apoptosis inhibitor or PPARγ agonist rosiglitazone prevented smoking-induced crypt apoptosis and Paneth cell defects in T300A mice and mice with conditional Paneth cell-specific knockout of Atg16l1. This study demonstrates how explicit G+E can drive disease-relevant phenotype and provides rational strategies for identifying actionable targets.