Single-cell multiomics guided mechanistic understanding of Fontan-associated liver disease.

The Fontan operation is the current standard of care for single-ventricle congenital heart disease. Individuals with a Fontan circulation (FC) exhibit central venous hypertension and face life-threatening complications of hepatic fibrosis, known as Fontan-associated liver disease (FALD). The fundamental biology and mechanisms of FALD are little understood. Here, we generated a transcriptomic and epigenomic atlas of human FALD at single-cell resolution using multiomic snRNA-ATAC-seq. We found profound cell type-specific transcriptomic and epigenomic changes in FC livers. Central hepatocytes (cHep) exhibited the most substantial changes, featuring profound metabolic reprogramming. These cHep changes preceded substantial activation of hepatic stellate cells and liver fibrosis, suggesting cHep as a potential first "responder" in the pathogenesis of FALD. We also identified a network of ligand-receptor pairs that transmit signals from cHep to hepatic stellate cells, which may promote their activation and liver fibrosis. We further experimentally demonstrated that activins A and B promote fibrotic activation in vitro and identified mechanisms of activin A's transcriptional activation in FALD. Together, our single-cell transcriptomic and epigenomic atlas revealed mechanistic insights into the pathogenesis of FALD and may aid identification of potential therapeutic targets.

Three-Dimensional Imaging of the Enteric Nervous System in Human Pediatric Colon Reveals New Features of Hirschsprung's Disease.

BACKGROUND & AIMS: Hirschsprung's disease is defined by the absence of the enteric nervous system (ENS) from the distal bowel. Primary treatment is "pull-through" surgery to remove bowel that lacks ENS, with reanastomosis of "normal" bowel near the anal verge. Problems after pull-through are common, and some may be due to retained hypoganglionic bowel (ie, low ENS density). Testing this hypothesis has been difficult because counting enteric neurons in tissue sections is unreliable, even for experts. Tissue clearing and 3-dimensional imaging provide better data about ENS structure than sectioning. METHODS: Regions from 11 human colons and 1 ileal specimen resected during Hirschsprung's disease pull-through surgery were cleared, stained with antibodies to visualize the ENS, and imaged by confocal microscopy. Control distal colon from people with no known bowel problems were similarly cleared, stained, and imaged. RESULTS: Quantitative analyses of human colon, ranging from 3 days to 60 years old, suggest age-dependent changes in the myenteric plexus area, ENS ganglion area, percentage of myenteric plexus occupied by ganglia, neurons/mm2, and neuron Feret's diameter. Neuron counting using 3-dimensional images was highly reproducible. High ENS density in neonatal colon allowed reliable neuron counts using 500-µm2 × 500-µm2 regions (36-fold smaller than in adults). Hirschsprung's samples varied 8-fold in proximal margin enteric neuron density and had diverse ENS architecture in resected bowel. CONCLUSIONS: Tissue clearing and 3-dimensional imaging provide more reliable information about ENS structure than tissue sections. ENS structure changes during childhood. Three-dimensional ENS anatomy may provide new insight into human bowel motility disorders, including Hirschsprung's disease.

Endoscopic and histologic utility of transnasal endoscopy in pediatric eosinophilic esophagitis.

Unsedated transnasal endoscopy (TNE) is an alternative method of examining the esophageal mucosa in pediatric patients with eosinophilic esophagitis (EoE), reducing cost, time, and risk associated with frequent surveillance esophagogastroduodenoscopies (EGD). Adequacy of transnasal esophageal biopsies for the evaluation of eosinophilic esophagitis histologic scoring system (EoEHSS) has not yet been evaluated. We compared procedure times, endoscopic findings, and EoEHSS scoring for EoE patients undergoing TNE versus standard EGD. Sixty-six TNE patients and 132 EGD controls matched for age (mean age 14.0 years) and disease status (29.3% active) were included. Compared to patients undergoing standard EGD, patients undergoing TNE spent 1.94 h less in the GI suite (p < 0.0001), with comparable occurrence rates of all visual endoscopic findings and most EoEHSS components. TNE serves as a useful tool for long-term disease surveillance, and consideration should be given to its use in clinical trials for EoE.

Gastrointestinal Tract Granular Cell Tumor in the Pediatric Population: A Multicenter Experience.

BACKGROUND: Pediatric granular cell tumors (GCT) involving the gastrointestinal tract (GIT) are rare with limited case report/series reported to date. METHODS: Multicenter retrospective study of pediatric GIT GCT. RESULTS: A total of 10 cases were included in the study with a median age of 13.5 years (range: 7-18 years) and were predominantly female patients (60%). In half of the patients no significant medical history was present with the remaining 5 having Crohn disease (10%), eosinophilic esophagitis (EoE) (10%), Crohn disease and EoE (10%), growth hormone deficiency (10%), and aplasia cutis congenita (10%). The GCT median size was 1.3 cm (range: 1-1.6 cm) and were more commonly located in the esophagus (70%) followed by the stomach (20%) and rectum (10%). Most of the cases showed round/polygonal tumor cells with abundant granular cytoplasm, and none of the cases had nuclear atypia, increased mitotic activity, or tumor cell necrosis. None of our cases received specific therapy for GCT other than clinical follow-up, and none of the patients had evidence of local recurrence or metastatic disease. CONCLUSION: We present our multicenter experience with GIT GCT, all cases had a benign course. Interestingly, 4 of the esophageal GCT cases (including 2 patients with EoE) showed an eosinophil-rich esophagitis in the underlying mucosa.

Rapid Quantification of Ammonium Nitrate and Urea Nitrate Using Liquid Chromatography-High-Resolution Orbitrap Mass Spectrometry.

Resolution and sensitivity improvements in mass spectrometry technology have enabled renewed attempts at solving challenging analytical issues. One such issue involves the analysis of energetic ionic species. Energetic ionic species make up an important class of chemical materials, and a more robust and versatile analytical platform would provide tremendous value to the analytical community. Initial attempts at quantification of energetic ionic species employed high-resolution time-of-flight measurements with crown ether (CE) complexation and flow injection analysis (FIA). In this investigation, ammonium nitrate (AN) and urea nitrate (UN) in the presence of a crown ether complexation agent were explored by using high-resolution orbitrap mass spectrometry. Product ion scans of these signature complexes reveal positive identification of these energetic ionic species. Finally, quantification was demonstrated for both flow injection and liquid chromatography-mass spectrometry (LC-MS) analysis, suggesting the capability for routine and rapid analysis of these energetic ionic materials.

Novel Development of Magnetic Resonance Imaging to Quantify the Structural Anatomic Growth of Diverse Organs in Adult and Mutant Zebrafish.

Zebrafish (Danio rerio) is a widely used vertebrate animal for modeling genetic diseases by targeted editing strategies followed by gross phenotypic and biomarker characterization. While larval transparency permits microscopic detection of anatomical defects, histological adult screening for organ-level defects remains invasive, tedious, inefficient, and subject to technical artifact. Here, we describe a noninvasive magnetic resonance imaging (MRI) approach to systematically screen adult zebrafish for anatomical growth defects. An anatomical atlas of wild-type (WT) zebrafish at 5-31 months post-fertilization was created by ex vivo MRI with a 9.4 T magnet. Volumetric growth over time was measured of animals and major organs, including the brain, spinal cord, heart, eyes, optic nerve, ear, liver, kidneys, and swim bladder. Subsequently, surf1-/-, fbxl4-/-, and opa1+/- mitochondrial disease mutant adult zebrafish were quantitatively studied to compare organ volumes with age-matched WT zebrafish. Results demonstrated that MRI enabled noninvasive, high-resolution, rapid screening of mutant adult zebrafish for overall and organ-specific growth abnormalities. Detailed volumetric analyses of three mitochondrial disease mutants delineated specific organ differences, including significantly increased brain growth in surf1-/- and opa1+/-, and marginally significant decreased heart and spinal cord volumes in surf1-/- mutants. This is interesting as we know neurological involvement can be seen in SURF1-/- patients with ataxia, dystonia, and lesions in basal ganglia, as well as in OPA1+/- patients with spasticity, ataxia, and hyperreflexia indicative of neuropathology. Similarly, cardiomyopathy is a known sequelae of cardiac pathology in patients with SURF1-/--related disease. Future studies will define MRI signaling patterns of organ dysfunction to further delineate specific pathology.

Intestinal transit-amplifying cells require METTL3 for growth factor signaling and cell survival.

Intestinal epithelial transit-amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite these cells' critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit-amplifying cell function. We report that RNA methyltransferase-like 3 (METTL3) is required for survival of transit-amplifying cells in the murine small intestine. Transit-amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Sequencing of polysome-bound and methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation verified a relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit-amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine with important implications for both homeostatic tissue renewal and epithelial regeneration.

Loss of zebrafish pkd1l1 causes biliary defects that have implications for biliary atresia splenic malformation.

Biliary atresia is a fibroinflammatory neonatal disease with no effective therapies. A subset of cases (10-20%) is associated with laterality defects - labeled biliary atresia splenic malformation (BASM) syndrome. Recently, whole-exome sequencing of patients with BASM identified deleterious variants in PKD1L1. PKD1L1 is involved in left-right axis determination; however, its role in cholangiocytes is unknown. We generated the pkd1l1hsc117 allele using CRISPR/Cas9 mutagenesis in zebrafish to determine the role of Pkd1l1 in biliary development and function. Wild-type and mutant larvae were assessed for laterality defects, biliary function and biliary tree architecture at 5 days post fertilization. pkd1l1hsc117 mutant larvae exhibited early left-right patterning defects. The gallbladder was positioned on the left in 47% of mutants compared to 4% of wild-type larvae. Accumulation of PED6 in the gallbladder, an indicator of hepatobiliary function, was significantly reduced in pkd1l1hsc117 mutants (46%) compared to wild-type larvae (4%). pkd1l1hsc117 larvae exhibited fewer biliary epithelial cells and reduced density of the intrahepatic biliary network compared to those in wild-type larvae. These data highlight the essential role of pkd1l1 in normal development and function of the zebrafish biliary system, supporting a role for this gene as a cause of BASM.

From the laboratory to the field: Chemical analysis of colored smoke pyrotechnic formulations via mass spectrometry techniques.

Smoke dyes are complex molecular systems that have the potential to form many molecular derivatives and fragments when deployed. The chemical analysis of smoke samples is challenging due to the adiabatic temperature of the pyrotechnic combustion and the molecular complexity of the physically dispersed reaction products. Presented here is the characterization of the reaction byproducts of a simulant Mk124 smoke signal on a multigram scale, which contain the dye disperse red 9 (1-(methylamino)anthraquinone), by ambient ionization mass spectrometry. Our previous work has examined the thermal decomposition of a simplified smoke system consisting of disperse red 9, potassium chlorate, and sucrose by anaerobic pyrolysis gas chromatography mass spectrometry performed at the laboratory milligram scale. The results from the lab scale test were compared with a fully functioned Mk124 in the field. To achieve this, Mk124 smokes were functioned in the presence of sampling swabs that collected byproduct residues from the smoke plume in the ambient environment. These swabs were then analyzed using ambient ionization mass spectrometry to identify the expended pyrotechnic residues, with particular interest in halogenated species. Previous work determined the toxicity of unforeseen byproducts identified on the laboratory scale, which were also detected in the field demonstrating the correlation of the laboratory testing to the fielded systems. By understanding the chemical composition of smokes and their reaction products, potential toxicity effects can be easily assessed, leading to safer formulations with improved performance. These results can help assess how smoke byproducts may impact Warfighter performance, personnel health, and the environment.

Intestinal transit amplifying cells require METTL3 for growth factor signaling, KRAS expression, and cell survival.

Intestinal epithelial transit amplifying cells are essential stem progenitors required for intestinal homeostasis, but their rapid proliferation renders them vulnerable to DNA damage from radiation and chemotherapy. Despite their critical roles in intestinal homeostasis and disease, few studies have described genes that are essential to transit amplifying cell function. We report that the RNA methyltransferase, METTL3, is required for survival of transit amplifying cells in the murine small intestine. Transit amplifying cell death after METTL3 deletion was associated with crypt and villus atrophy, loss of absorptive enterocytes, and uniform wasting and death in METTL3-depleted mice. Ribosome profiling and sequencing of methylated RNAs in enteroids and in vivo demonstrated decreased translation of hundreds of unique methylated transcripts after METTL3 deletion, particularly transcripts involved in growth factor signal transduction such as Kras. Further investigation confirmed a novel relationship between METTL3 and Kras methylation and protein levels in vivo. Our study identifies METTL3 as an essential factor supporting the homeostasis of small intestinal tissue via direct maintenance of transit amplifying cell survival. We highlight the crucial role of RNA modifications in regulating growth factor signaling in the intestine, with important implications for both homeostatic tissue renewal and epithelial regeneration.

No-Implant Interatrial Shunt for HFpEF: 6-Month Outcomes From Multicenter Pilot Feasibility Studies.

BACKGROUND: Most approaches to the creation of an interatrial shunt require placement of a permanent implant to maintain patency. OBJECTIVES: The goal of this study was to investigate the safety and efficacy of a no-implant interatrial shunt for patients with heart failure with preserved ejection fraction (HFpEF) and heart failure with mildly reduced ejection fraction (HFmrEF). METHODS: This was a multicenter, uncontrolled study of patients with HFpEF/HFmrEF and NYHA functional class ≥II, ejection fraction >40%, and pulmonary capillary wedge pressure (PCWP) during supine exercise ≥25 mm Hg with PCWP-to-right atrial gradient ≥5 mm Hg. Follow-up was through 6 months with imaging to assess shunt durability. RESULTS: A total of 28 patients were enrolled: mean age was 68 ± 9 years, and 68% were female. Baseline resting and peak exercise PCWP were 19 ± 7 mm Hg and 40 ± 11 mm Hg, respectively. All procedures displayed technical success with confirmation of left-to-right flow (shunt diameter 7.1 ± 0.9 mm). At 1 month, peak exercise PCWP decreased 5.4 ± 9.6 mm Hg (P = 0.011) with no change in right atrial pressure. There were no serious device or procedure-related adverse events through 6 months. Mean 6-minute walk distance increased 101 ± 71 meters (P < 0.001); Kansas City Cardiomyopathy Questionnaire Overall Summary Score increased 26 ± 19 points (P < 0.001); N-terminal pro-B-type natriuretic peptide decreased 372 ± 857 pg/mL (P = 0.018); and shunt patency was confirmed with unchanged diameter. CONCLUSIONS: In these feasibility studies of a no-implant interatrial shunt, HFpEF/HFmrEF shunts exhibited stability with favorable safety and early efficacy signals. The results show promise toward this new approach for treating patients with HFpEF/HFmrEF and an appropriate hemodynamic profile. (Evaluation of the Safety and Feasibility of a Percutaneously Created Interatrial Shunt to Alleviate Heart Failure Symptoms in Patients With Chronic Heart Failure and Preserved or Mid-Range Left Ventricular Ejection Fraction [ALLEVIATE-HF-1]; NCT04583527; Evaluation of the Safety and Effectiveness of a Percutaneously Created Interatrial Shunt to Alleviate Heart Failure Symptoms in Patients With Chronic Heart Failure and Preserved or Mid-Range Left Ventricular Ejection Fraction [ALLEVIATE-HF-2]; NCT04838353).

The poly(C)-binding protein Pcbp2 is essential for CD4+ T cell activation and proliferation.

The RNA-binding protein Pcbp2 is widely expressed in the innate and adaptive immune systems and is essential for mouse development. To determine whether Pcbp2 is required for CD4+ T cell development and function, we derived mice with conditional Pcbp2 deletion in CD4+ T cells and assessed their overall phenotype and proliferative responses to activating stimuli. We found that Pcbp2 is essential for T conventional cell (Tconv) proliferation, working through regulation of co-stimulatory signaling. Pcbp2 deficiency in the CD4+ lineage did not impact Treg abundance in vivo or function in vitro. In addition, our data demonstrate a clear association between Pcbp2 control of Runx1 exon 6 splicing in CD4+ T cells and a specific role for Pcbp2 in the maintenance of peripheral CD4+ lymphocyte population size. Last, we show that Pcbp2 function is required for optimal in vivo Tconv cell activation in a T cell adoptive transfer colitis model system.

The Slender Esophagus: Unrecognized Esophageal Narrowing in Eosinophilic Esophagitis.

INTRODUCTION: Inflammation in eosinophilic esophagitis (EoE) often leads to esophageal strictures. Evaluating esophageal narrowing is clinically challenging. We evaluated esophageal distensibility as related to disease activity, fibrosis, and dysphagia. METHODS: Adult patients with and without EoE underwent endoscopy and distensibility measurements. Histology, distensibility, and symptoms were analyzed. RESULTS: Patients with EoE had significantly lower distensibilities than controls. We found a cohort with esophageal diameter under 15 mm despite lack of dysphagia. DISCUSSION: This study raises concern that current assessments of fibrostenosis are suboptimal. We describe a cohort with unrecognized slender esophagus that were identified through impedance planimetry measurements. This tool provides additional information beyond symptomatic, histologic, and endoscopic assessments.

Validity and feasibility of remote measurement systems for functional movement and posture assessments in people with axial spondylarthritis.

INTRODUCTION: This study aimed to estimate the criterion validity of functional movement and posture measurement using remote technology systems in people with and without Axial spondylarthritis (axSpA). METHODS: Validity and agreement of the remote-technology measurement of functional movement and posture were tested cross-sectionally and compared to a standard clinical measurement by a physiotherapist. The feasibility of remote implementation was tested in a home environment. There were two cohorts of participants: people with axSpA and people without longstanding back pain. In addition, a cost-consequence analysis was performed. RESULTS: Sixty-two participants (31 with axSPA, 53% female, age = 45(SD14), BMI = 26.6(SD4.6) completed the study. In the axSpA group, cervical rotation, lumbar flexion, lumbar side flexion, shoulder flexion, hip abduction, tragus-to-wall and thoracic kyphosis showed a significant moderate to strong correlation; in the non-back pain group, the same measures showed significant correlation ranging from weak to strong. CONCLUSIONS: Although not valid for clinical use in its current form, the remote technologies demonstrated moderate to strong correlation and agreement in most functional and postural tests measured in people with AxSA. Testing the CV-aided system in a home environment suggests it is a safe and feasible method. Yet, validity testing in this environment still needs to be performed.

Geometric engineering of organoid culture for enhanced organogenesis in a dish.

Here, we introduce a facile, scalable engineering approach to enable long-term development and maturation of organoids. We have redesigned the configuration of conventional organoid culture to develop a platform that converts single injections of stem cell suspensions to radial arrays of organoids that can be maintained for extended periods without the need for passaging. Using this system, we demonstrate accelerated production of intestinal organoids with significantly enhanced structural and functional maturity, and their continuous development for over 4 weeks. Furthermore, we present a patient-derived organoid model of inflammatory bowel disease (IBD) and its interrogation using single-cell RNA sequencing to demonstrate its ability to reproduce key pathological features of IBD. Finally, we describe the extension of our approach to engineer vascularized, perfusable human enteroids, which can be used to model innate immune responses in IBD. This work provides an immediately deployable platform technology toward engineering more realistic organ-like structures in a dish.

Solid-Tubulocystic Variant of Intrahepatic Cholangiocarcinoma: Report of a Pediatric Case With Molecular Characterization.

We present a case of solid-tubulocystic variant of intrahepatic cholangiocarcinoma (also called cholangioblastic variant of intrahepatic cholangiocarcinoma) in a 15-year-old girl, the youngest patient reported to date. The tumor was located in the left lobe of the liver, predominantly solid with cystic areas, and measured 16 cm in greatest dimension. Microscopic examination showed 2 major histologic patterns: a mixed pattern with solid, tubulocystic, macrocystic, trabecular, and nested growth, diffuse cytokeratin 7/19 and weak neuroendocrine immunoreactivity, and low Ki-67 index; and a more compact, macrotrabecular/gyriform pattern with focal CK7/19, stronger neuroendocrine reactivity, and higher Ki-67 index. Inhibin immunoreactivity was diffuse throughout both patterns. Treatment included tumor resection with negative margins and 8 cycles of capecitabine chemotherapy; the patient is alive with no evidence of tumor 2.5 years after resection. Although molecular characterization of the tumor at the time of resection was unrevealing, a recent study has identified a novel NIPBL-NACC1 fusion transcript in this tumor type, which we have confirmed in this case. This case expands the reported age range of this rare tumor type and confirms a recently-reported diagnostic genomic alteration. Awareness of this rare entity affecting pediatric patients is crucial to avoid confusion with similar-appearing neoplasms.

Esophageal remodeling in eosinophilic esophagitis: Relationships to luminal captured biomarkers of inflammation and periostin.

BACKGROUND: Esophageal remodeling is a factor in disease progression and symptom severity for patients with eosinophilic esophagitis (EoE). Remodeling can begin early in children, resulting in stricture and food impaction. Detection of esophageal remodeling often depends on endoscopy and is appreciated only in its later stages. OBJECTIVE: We sought to determine whether luminal eosinophil-associated and remodeling proteins captured by the esophageal string test (EST) correlate with measures of esophageal remodeling and biomarkers of the epithelial-mesenchymal transition (EMT). METHODS: Patients with EoE (7-18 years old) were enrolled from 2 pediatric hospitals. Participants performed the EST and underwent endoscopy. Histology, distensibility measured by endoluminal functional lumen imaging probe, and symptoms were assessed. Protein quantitation by ELISA was performed on mucosal biopsy and EST samples. Tissue sections were evaluated for EMT. Outcome measures were summarized, and Spearman ρ was used to assess bivariate correlations. RESULTS: Forty patients (68% male) were enrolled (mean age, 12.5 years). Twenty-four (60%) had active disease (≥15 eosinophils per high-power field). EST-captured eotaxin-3, major basic protein 1, EDN, eosinophil peroxidase, and Charcot-Leyden crystal protein/galectin-10 showed significant correlations with peak eosinophils per high-power field (ρ 0.53-0.68, P < .001). Luminal proteins positively correlated with endoscopic features and markers of EMT, and negatively with esophageal distensibility. Periostin was captured by the EST and correlated with eosinophil density, basal zone hyperplasia, endoscopic appearance, and markers of EMT. CONCLUSION: Luminal markers of esophageal remodeling in addition to biomarkers of eosinophilic inflammation correlate with epithelial and functional remodeling in EoE.

CD73+ Epithelial Progenitor Cells That Contribute to Homeostasis and Renewal Are Depleted in Eosinophilic Esophagitis.

BACKGROUND & AIMS: Although basal cell hyperplasia is a histologic hallmark of eosinophilic esophagitis (EoE), little is known about the capabilities of epithelial renewal and differentiation in the EoE inflammatory milieu. In murine esophageal epithelium, there are self-renewing and slowly proliferating basal stem-like cells characterized by concurrent expression of CD73 (5'-nucleotidase ecto) and CD104 (integrin ß4). Here, we investigated CD73+CD104+ cells within the basal population of human esophageal epithelium and clarified the biological significance of these cells in the EoE epithelium. METHODS: We performed flow cytometry on esophageal biopsy samples from EoE and non-EoE patients to determine the quantity of CD73+CD104+ cells in the epithelium. Simulating the EoE milieu we stimulated primary patient-derived and immortalized cell line-derived esophageal organoids with interleukin (IL)4 and IL13 and analyzed by flow cytometry, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. We performed single-cell RNA sequencing on primary organoids in the setting of IL13 stimulation and evaluated the CD73+CD104+ population. We performed fluorescent-activated cell sorting to purify CD73+CD104+ and CD73- CD104+ populations and seeded these groups in organoid culture to evaluate the organoid formation rate and organoid size. We used RNA interference to knock down CD73 in esophageal organoids to evaluate organoid formation rates and size. We evaluated the effects of signal transducer and activator of transcription 6 (STAT6) signaling inhibition by RNA interference, a STAT6 inhibitor, AS1517499, as well as the proton pump inhibitor omeprazole. RESULTS: EoE patients showed decreased epithelial CD73+CD104+ cell content. IL4 and IL13 stimulation depleted this population in 3-dimensional organoids with a recapitulation of basal cell hyperplasia as corroborated by single-cell RNA sequencing of the organoids, which suggests depletion of CD73+CD104+ cells. The CD73+CD104+ population had enhanced organoid formation compared with the CD73-CD104+ population. Similarly, knock-down of CD73 resulted in decreased organoid formation rate. Genetic and pharmacologic inhibition of STAT6 prevented T helper 2 cytokine-induced depletion of CD73+CD104+ cells. Lastly, omeprazole treatment prevented the effects of IL4 and IL13 on the CD73+CD104+ population. CONCLUSIONS: This study addressed the role of CD73+CD104+ cells in epithelial renewal and homeostasis in the context of EoE. The depletion of the CD73+CD104+ self-renewal population by helper T cell 2 cytokines in EoE milieu may be perpetuating epithelial injury. Future therapies targeting epithelial restitution in EoE could decrease the need for immune modulation and steroid therapy.

Detection and toxicity modeling of anthraquinone dyes and chlorinated side products from a colored smoke pyrotechnic reaction.

"Green" pyrotechnics seek to remove known environmental pollutants and health hazards from their formulations. This chemical engineering approach often focuses on maintaining performance effects upon replacement of objectionable ingredients, yet neglects the chemical products formed by the exothermic reaction. In this work, milligram quantities of a lab-scale pyrotechnic red smoke composition were functioned within a thermal probe for product identification by pyrolysis-gas chromatography-mass spectrometry. Thermally decomposed ingredients and new side product derivatives were identified at lower relative abundances to the intact organic dye (as the engineered sublimation product). Side products included chlorination of the organic dye donated by the chlorate oxidizer. Machine learning quantitative structure-activity relationship models computed impacts to health and environmental hazards. High to very high toxicities were predicted for inhalation, mutagenicity, developmental, and endocrine disruption for common military pyrotechnic dyes and their analogous chlorinated side products. These results underscore the need to revise objectives of "green" pyrotechnic engineering.