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Breaking codon degeneracy for the introduction of non-canonical amino acids offers many opportunities in synthetic biology. Yet, despite the existence of 64 codons, the code has only been expanded to 25 amino acids in vitro. A limiting factor could be the over-reliance on synthetic tRNAs which lack the post-transcriptional modifications that improve translational fidelity. To determine whether modified, wild-type tRNA could improve sense codon reassignment, we developed a new fluorous method for tRNA capture and applied it to the isolation of roughly half of the Escherichia coli tRNA isoacceptors. We then performed codon competition experiments between the five captured wild-type leucyl-tRNAs and their synthetic counterparts, revealing a strong preference for wild-type tRNA in an in vitro translation system. Finally, we compared the ability of wild-type and synthetic leucyl-tRNA to break the degeneracy of the leucine codon box, showing that only captured wild-type tRNAs are discriminated with enough fidelity to accurately split the leucine codon box for the encoding of three separate amino acids. Wild-type tRNAs are therefore enabling reagents for maximizing the reassignment potential of the genetic code.

The discovery of high-affinity peptides to many intracellular targets has become feasible through the development of diverse macrocyclic peptide libraries. But lack of cell permeability is a key feature hampering the use of these peptides as therapeutics. Here, we develop a set of small, cyclic peptide carriers that efficiently carry cargoes into the cytosol. These peptides are cyclized via side-chain alkylation, which makes them ideal for the creation of diverse mRNA or phage-displayed libraries with intrinsic cell permeability.

The ability to introduce non-canonical amino acids into peptides and proteins is facilitated by working within in vitro translation systems. Non-canonical amino acids can be introduced into these systems using sense codon reprogramming, stop codon suppression, and by breaking codon degeneracy. Here, we review how these techniques have been used to create proteins with novel properties and how they facilitate sophisticated studies of protein function. We also discuss how researchers are using in vitro translation experiments with non-canonical amino acids to explore the tolerance of the translation apparatus to artificial building blocks. Finally, we give several examples of how non-canonical amino acids can be combined with mRNA-displayed peptide libraries for the creation of protease-stable, macrocyclic peptide libraries for ligand discovery.

The ability to incorporate non-canonical amino acids (ncAA) using translation offers researchers the ability to extend the functionality of proteins and peptides for many applications including synthetic biology, biophysical and structural studies, and discovery of novel ligands. Here we describe the high promiscuity of an editing-deficient valine-tRNA synthetase (ValRS T222P). Using this enzyme, we demonstrate ribosomal translation of 11 ncAAs including those with novel side chains, α,α-disubstitutions, and cyclic β-amino acids.

Hydrocarbon stapled peptides are promising therapeutics for inhibition of intracellular protein-protein interactions. Here we develop a new high-throughput strategy for hydrocarbon stapled peptide discovery based on mRNA display of peptides containing α-methyl cysteine and cyclized with m-dibromoxylene. We focus on development of a peptide binder to the HPV16 E2 protein.

Objectives: Eosinophilic esophagitis (EoE) is an immune-mediated allergic disease characterized by progressive esophageal dysmotility and fibrotic stricture associated with chronic esophageal fibroblast activation. It remains unknown how esophageal fibroblasts respond to EoE-relevant matrix stiffness or inflammatory cytokines. Methods: Immunofluorescence was used to evaluate α-smooth muscle actin (α-SMA) expression in endoscopic esophageal biopsies. Primary esophageal fibroblasts from adult and pediatric patients with or without EoE were exposed to transforming growth factor (TGF)β to determine gene expression, collagen-matrix contractility, and cytoskeletal organization. The influence of matrix stiffness upon fibroblast behavior was assessed on the engineered surface of polyacrylamide gels with varying stiffness. Fibroblast traction forces were measured using microfabricated-post-array-detectors. Results: EoE esophageal fibroblasts had enhanced α-SMA expression. TGFβ not only stimulated enhanced fibroblast-specific gene expression but also promoted fibroblast-mediated collagen-matrix contraction, despite disease state or age of patients as the origin of cells. Unlike conventional monolayer cell, culture conditions using plastic surface (1 GPa) that activates fibroblasts constitutively, our engineered platforms recapitulating physiologically relevant stiffness (1–20 kPa) revealed that matrix stiffness defines the extent of α-SMA expression, intracellular collagen fibril organization, SMAD3 phosphorylation, and fibroblast traction force. Conclusions: Matrix stiffness may critically influence TGFβ-mediated gene expression and functions of esophageal fibroblasts ex vivo independent of age and disease conditions. These findings provide a novel insight into the pathogenesis of fibrostenotic disease in EoE.

The prevalence of allergic conditions has continuously increased in the last few decades in Westernized countries. A dysbiotic gut microbiome may play an important role in the development of allergic diseases. Genetic, environmental, and dietary factors may alter the commensal microbiota leading to inflammatory dysregulation of homeostasis. Murine and human studies have begun to elucidate the role of the microbiota in the pathogenesis of atopic diseases including asthma, atopic dermatitis, and food allergies. However, the role of the microbiome in most eosinophilic gastrointestinal diseases (EGIDs) is not yet known. This review provides an overview of what is currently known about the development of tolerance from both molecular and clinical standpoints. We also look at the gut-specific microbiome and its role in atopic conditions with the hope of applying this knowledge to the understanding, prevention, and treatment of EGIDs, particularly EoE.

Eosinophilic esophagitis (EoE) is a chronic Th2 and food antigen-mediated disease characterized by esophageal eosinophilic infiltration. Thymic stromal lymphopoetin (TSLP), an epithelial derived cytokine which bridges innate and Th2-type adaptive immune responses in other allergic conditions, is overexpressed in esophageal biopsies of EoE subjects. However, the triggers of TSLP expression in the esophageal epithelium are unknown. The objective of the current study was to characterize TSLP expression in human esophageal epithelium in EoE in vivo and to determine the role of food antigens upon epithelial TSLP expression in vitro. Using immunohistochemistry (IHC), we localized TSLP in esophageal biopsies of active EoE (≥15 eos/hpf), inactive EoE (

Objective The influence of eosinophilic oesophagitis (EoE)-associated inflammation upon oesophageal epithelial biology remains poorly understood. We investigated the functional role of autophagy in oesophageal epithelial cells (keratinocytes) exposed to the inflammatory EoE milieu. Design Functional consequences of genetic or pharmacological autophagy inhibition were assessed in endoscopic oesophageal biopsies, human oesophageal keratinocytes, single cell-derived ex vivo murine oesophageal organoids as well as a murine model recapitulating EoE-like inflammation and basal cell hyperplasia. Gene expression, morphological and functional characterisation of autophagy and oxidative stress were performed by transmission electron microscopy, immunostaining, immunoblotting, live cell imaging and flow cytometry. Results EoE-relevant inflammatory conditions promoted autophagy and basal cell hyperplasia in three independent murine EoE models and oesophageal organoids. Inhibition of autophagic flux via chloroquine treatment augmented basal cell hyperplasia in these model systems. Oesophageal keratinocytes stimulated with EoE-relevant cytokines, including tumour necrosis factor-α and interleukin-13 exhibited activation of autophagic flux in a reactive oxygen species-dependent manner. Autophagy inhibition via chloroquine treatment or depletion of Beclin-1 or ATG-7, augmented oxidative stress induced by EoE-relevant stimuli in murine EoE, oesophageal organoids and human oesophageal keratinocytes. Oesophageal epithelia of paediatric EoE patients with active inflammation displayed increased autophagic vesicle content compared with normal and EoE remission subjects. Functional flow cytometric analysis revealed autophagic flux in human oesophageal biopsies. Conclusions Our findings reveal for the first time that autophagy may function as a cytoprotective mechanism to maintain epithelial redox balance and homeostasis under EoE inflammation-associated stress, providing mechanistic insights into the role of autophagy in EoE pathogenesis.

Eosinophilic Esophagitis (EoE) is a chronic allergic disorder, whose pathobiology is incompletely understood. Histamine-producing cells including mast cells and basophils have been implicated in EoE. However, very little is currently known about the role of histamine and histamine receptor (HR) expression and signaling in the esophageal epithelium. Herein, we characterized HR (H1R, H2R, H3R, and H4R) expression in human esophageal biopsies and investigate the role of histamine signaling in inducible cytokine expression in human esophageal epithelial cells in vitro. HR expression was quantified in esophageal biopsies from non-EoE control (N = 23), inactive EoE (15 eos/hpf, N = 22) subjects using qRT-PCR and immunofluorescent localization. HR expression and histamine-mediated cytokine secretion were evaluated in human primary and telomerase-immortalized esophageal epithelial cells. H1R, H2R, and H4R expression were increased in active EoE biopsies compared to inactive EoE and controls. H2R was the most abundantly expressed receptor, and H3R expression was negligible in all 3 cohorts. Infiltrating eosinophils expressed H1R, H2R, and H4R, which contributed to the observed increase in HR in active subjects. H1R and H2R, but not H3R or H4R, were constitutively expressed by primary and immortalized cells, and epithelial histamine stimulation induced GM-CSF, TNFα, and IL-8, but not TSLP or eotaxin-3 secretion. Epithelial priming with the TLR3 ligand poly (I:C) induced H1R and H2R expression, and enhanced histamine-induced GM-CSF, TNFα, and IL-8 secretion. These effects were primarily suppressed by H1R antagonists, but unaffected by H2R antagonism. Histamine directly activates esophageal epithelial cytokine secretion in vitro in an H1R dependent fashion. However, H1R, H2R and H4R are induced in active inflammation in EoE in vivo. While systemic antihistamine (anti-H1R) therapy may not induce clinical remission in EoE, our study suggests that further study of histamine receptor signaling in EoE is warranted and that targeting of additional histamine receptors may lead to novel treatment strategies for this important disease.

Eosinophilic esophagitis (EoE) is an allergic inflammatory disease that leads to esophageal fibrosis and stricture. We have recently shown that in EoE, esophageal epithelial cells undergo an epithelial to mesenchymal transition (EMT), characterized by gain of mesenchymal markers and loss of epithelial gene expression. Whether epithelial cells exposed to profibrotic cytokines can also acquire the functional characteristics of activated myofibroblasts, including migration, contraction, and extracellular matrix deposition, is relevant to our understanding and treatment of EoE-associated fibrogenesis. In the current study, we characterize cell migration, contraction, and collagen production by esophageal epithelial cells that have undergone cytokine-induced EMT in vitro.Stimulation of human non-transformed immortalized esophageal epithelial cells (EPC2-hTERT) with profibrotic cytokines TNFα, TGFβ, and IL1β for three weeks led to acquisition of mesenchymal αSMA and vimentin, and loss of epithelial E-cadherin expression. Upon removal of the profibrotic stimulus, epithelial characteristics were partially rescued. TGFβ stimulation had a robust effect upon epithelial collagen production. Surprisingly, TNFα stimulation had the most potent effect upon cell migration and contraction, exceeding the effects of the prototypical profibrotic cytokine TGFβ. IL1β stimulation alone had minimal effect upon esophageal epithelial migration, contraction, and collagen production.Esophageal epithelial cells that have undergone EMT acquire functional characteristics of activated myofibroblasts in vitro. Profibrotic cytokines exert differential effects upon esophageal epithelial cells, underscoring complexities of fibrogenesis in EoE, and implicating esophageal epithelial cells as effector cells in EoE-associated fibrogenesis.

of SBI in a pediatric patient with an acute diarrhea due to SIBO and/or antibiotic therapy. SBI may prove to be useful in some acute setting where limited therapeutic options are available. Given that SBI has been designated as a medical food with GRAS (General Recognized As Safe) status for use in the general population, it may serve as an option for children in the nutritional management of chronic loose and frequent stools.

Background Eosinophilic esophagitis (EoE) is an allergic disorder characterized by eosinophil-predominant esophageal inflammation, which can be ameliorated by food antigen restriction. Though recent studies suggest that changes in dietary composition may alter the distal gut microbiome, little is currently known about the impact of a restricted diet upon microbial communities of the oral and esophageal microenvironments in the context of EoE. We hypothesize that the oral and esophageal microbiomes of EoE patients are distinct from non-EoE controls, that these differences correspond to changes in esophageal inflammation, and that targeted therapeutic dietary intervention may influence community structure. Using 16S rRNA gene sequencing, we characterized the bacterial composition of the oral and esophageal microenvironments using oral swabs and esophageal biopsies from 35 non-EoE pediatric controls and compared this cohort to samples from 33 pediatric EoE subjects studied in a longitudinal fashion before and after defined dietary changes. Results Firmicutes were more abundant in esophageal samples compared to oral. Proportions of bacterial communities were significantly different comparing all EoE esophageal microbiota to non-EoE controls, with enrichment of Proteobacteria, including Neisseria and Corynebacterium in the EoE cohort, and predominance of the Firmicutes in non-EoE control subjects. We detected a statistically significant difference between actively inflamed EoE biopsies and non-EoE controls. Overall, though targeted dietary intervention did not lead to significant differences in either oral or esophageal microbiota, reintroduction of highly allergenic foods led to enrichment in Ganulicatella and Campylobacter genera in the esophagus. Conclusions In conclusion, the esophageal microbiome in EoE is distinct from that of non-EoE controls, with maximal differences observed during active allergic inflammation. Electronic supplementary material The online version of this article (doi:10.1186/s40168-015-0085-6) contains supplementary material, which is available to authorized users.