Background: Assessment of approved drugs and developmental drug candidates for rare cystic fibrosis (CF)-causing variants of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) requires abundant material from relevant models. Methods: Isogenic cell lines harboring CFTR variants in the native genomic context were created through the development and utilization of a footprint-less, CRISPR/Cas9 gene editing pipeline in 16HBE14o-immortalized bronchial epithelial cells. Results: Isogenic, homozygous cell lines for three CFTR variants (F508del and the two most common CF-causing nonsense variants, G542X and W1282X) were established and characterized. The F508del model recapitulates the known molecular pathology and pharmacology. The two models of nonsense variants (G542X and W1282X) are sensitive to Nonsense Mediated mRNA Decay (NMD) and responsive to reference compounds that inhibit NMD and promote ribosomal readthrough. Conclusions: We present a versatile, efficient gene editing pipeline that can be used to create CFTR variants in the native genomic context and the utilization of this pipeline to create homozygous cell models for the CF-causing variants F508del, G542X, and W1282X. The resulting cell lines provide a virtually unlimited source of material with specific pathogenic mutations that can be used in a variety of assays, including functional assays.

The epithelial Na+ channel (ENaC) that mediates regulated Na+ reabsorption by epithelial cells in the kidney and lungs can be activated by endogenous proteases such as channel activating protease 1 and exogenous proteases such as trypsin and neutrophil elastase (NE). The mechanism by which exogenous proteases activate the channel is unknown. To test the hypothesis that residues on ENaC mediate protease-dependent channel activation wild-type and mutant ENaC were stably expressed in the FRT epithelial cell line using a tripromoter human ENaC construct, and protease-induced short-circuit current activation was measured in aprotinin-treated cells. The amiloride-sensitive short circuit current (INa) was stimulated by aldosterone (1.5-fold) and dexamethasone (8-fold). Dexamethasone-treated cells were used for all subsequent studies. The serum protease inhibitor aprotinin decreased baseline INa by approximately 50% and INa could be restored to baseline control values by the exogenous addition of trypsin, NE, and porcine pancreatic elastase (PE) but not by thrombin. All protease experiments were thus performed after exposure to aprotinin. Because NE recognition of substrates occurs with a preference for binding valines at the active site, several valines in the extracellular loops of α and γ ENaC were sequentially substituted with glycines. This scan yielded two valine residues in γ ENaC at positions 182 and 193 that resulted in inhibited responses to NE when simultaneously changed to other amino acids. The mutations resulted in decreased rates of activation and decreased activated steady-state current levels. There was an ∼20-fold difference in activation efficiency of NE against wild-type ENaC compared to a mutant with glycine substitutions at positions 182 and 193. However, the mutants remain susceptible to activation by trypsin and the related elastase, PE. Alanine is the preferred P1 position residue for PE and substitution of alanine 190 in the γ subunit eliminated INa activation by PE. Further, substitution with a novel thrombin consensus sequence (LVPRG) beginning at residue 186 in the γ subunit (γTh) allowed for INa activation by thrombin, whereas wild-type ENaC was unresponsive. MALDI-TOF mass spectrometric evaluation of proteolytic digests of a 23-mer peptide encompassing the identified residues (T176-S198) showed that hydrolysis occurred between residues V193 and M194 for NE and between A190 and S191 for PE. In vitro translation studies demonstrated thrombin cleaved the γTh but not the wild-type γ subunit. These results demonstrate that γ subunit valines 182 and 193 are critical for channel activation by NE, alanine 190 is critical for channel activation by PE, and that channel activation can be achieved by inserting a novel thrombin consensus sequence. These results support the conclusion that protease binding and perhaps cleavage of the γ subunit results in ENaC activation.

Autism spectrum disorder (ASD) is a severe brain development disorder that is characterized by deficits in social communication and restricted, repetitive and stereotyped behaviors. Accumulating evidence has suggested that gut microbiota disorders play important roles in gastrointestinal symptoms and neurodevelopmental dysfunction in ASD patients. Manipulation of the gut microbiota by fecal microbiota transplantation (FMT) was recently shown to be a promising therapy for the treatment of various diseases. Here, we performed a clinical trial to evaluate the effect of FMT on gastrointestinal (GI) and ASD symptoms and gut microbiota alterations in children with ASD. We found that there was a large difference in baseline characteristics of behavior, GI symptoms, and gut microbiota between children with ASD and typically developing (TD) control children. FMT could improve GI symptoms and ASD symptoms without inducing any severe complications. Similarly, FMT significantly changed the serum levels of neurotransmitters. We further observed that FMT could promote the colonization of donor microbes and shift the bacterial community of children with ASD toward that of TD controls. The abundance of Eubacterium coprostanoligenes pre-FMT was positively correlated with high GSRS scores, whereas a decrease in Eubacterium coprostanoligenes abundance induced by FMT was associated with the FMT response. Our data suggest that FMT might be a promising therapeutic strategy to improve the GI and behavioral symptoms of patients with ASD, possibly due to its ability to alter gut microbiota and highlight a specific microbiota intervention that targets Eubacterium coprostanoligenes that can enhance the FMT response. This trial was registered at the Chinese Clinical Trial Registry (www.chictr.org.cn) (trial registration number ChiCTR1800014745).

Accumulating evidence suggests a role of bisphenol A (BPA) in metabolic disorders. However, the underlying mechanism is still unclear. Using a mouse BPA exposure model, we investigated the effects of long-term BPA exposure on lipid metabolism and the underlying mechanisms. The male mice exposed to BPA (0.5 μg BPA /kg/day, a human relevant dose) for 10 months exhibited significant hepatic accumulation of triglycerides and cholesterol. The liver cells from the BPA-exposed mice showed significantly increased expression levels of the genes related to lipid synthesis. These liver cells showed decreased DNA methylation levels of Srebf1 and Srebf2, and increased expression levels of Srebf1 and Srebf2 that may upregulate the genes related to lipid synthesis. The expression levels of DNA methyltransferases were decreased in BPA-exposed mouse liver. Hepa1-6 cell line treated with BPA showed decreased expression levels of DNA methyltransferases and increased expression levels of genes involved in lipid synthesis. DNA methyltransferase knockdown in Hepa1-6 led to hypo-methylation and increased expression levels of genes involved in lipid synthesis. Our results suggest that long-term BPA exposure could induce hepatic lipid accumulation, which may be due to the epigenetic reprogramming of the genes involved in lipid metabolism, such as the alterations of DNA methylation patterns.