GLUT1 mediates bronchial epithelial E-cadherin disruption in TDI-induced steroid-insensitive asthma.

OBJECTIVE: Down-regulation of bronchial epithelial E-cadherin is an important of feature of severe asthma, including steroid-insensitive asthma. Yet, the mechanisms involved in E-cadherin disruption are not fully understood. This study was aimed to investigate the role of glucose transporter 1 (GLUT1) in dysregulation of E-cadherin in toluene diisocyanate (TDI)-induced steroid-insensitive asthma. METHODS: A murine model of steroid-insensitive asthma was established by TDI sensitization and aerosol inhalation. Selective GLUT1 antagonists WZB117 and BAY876 were given to BALB/c mice after airway challenge. In vitro, primary human bronchial epithelial cells (HBECs) cultured in an airway-liquid interface (ALI) were exposed to TDI. RESULTS: TDI exposure markedly up-regulated GLUT1 in murine lungs and HBECs. Pharmacological inhibition of GLUT1 with BAY876 decreased airway hyperresponsiveness, neutrophil and eosinophil accumulation, as well as type 2 inflammation in vivo. Besides, the TDI-induced down-regulated expression of full-length E-cadherin was also partly recovered, accompanied by inhibited secretion of soluble E-cadherin (sE-cadherin). WZB117 also exhibited mild therapeutic effects, though not significant. In vitro, treatment with GLUT1 inhibitor relieved the TDI-induced disruption of E-cadherin in HBECs. CONCLUSIONS: Taken together, our data demonstrated that GLUT1 modulates bronchial epithelial E-cadherin dysfunction production in TDI-induced steroid-insensitive asthma.

Identification of soybean mosaic virus strain SC7 resistance loci and candidate genes in soybean [Glycine max (L.) Merr.].

Soybean [Glycine max (L.) Merr.] is an important legume crop worldwide, which provides abundant plant protein and oil for human beings. Soybean mosaic virus (SMV) can cause serious damage to the yield and quality of soybean, but it is difficult to control SMV with chemicals, breeding SMV-resistant varieties has become the most effective way to control the disease. Therefore, it is important to identify SMV resistance genes from soybean resources and apply them to soybean breeding. In this study, the disease rates (DRs) of 219 soybean accessions to SMV strain SC7 in two environments were investigated. A high-density NJAU 355 K SoySNP array was used for genome-wide association study (GWAS) of DR. A 274 kb region on chromosome 15 (1,110,567 bp to 1,384,173 bp) was repeatedly detected in two environments. Six new significant single nucleotide polymorphisms (SNPs) on chromosome 15 were identified. Four of these six SNPs were located within two candidate genes, Glyma.15G015700 and Glyma.15G015800. The elite haplotype Glyma.15G015700Hap I with low DR exhibited strong resistance to SC7. The expression of Glyma.15G015700 in the SMV-resistant accession increased significantly after inoculation with SC7. Furthermore, most of the proteins predicted to interact with Glyma.15G015700 are heat shock proteins, which have been shown to be related to disease resistance. In summary, new SMV resistance loci and a new candidate gene, Glyma.15G015700, were identified and might be utilized in further soybean disease resistance breeding.

Cytochrome GmGLY1 is Involved in the Biosynthesis of Glycitein in Soybean.

Isoflavones, the major secondary metabolites of interest due to their benefits to both human and plant health, are exclusively produced by legumes. In this study, we profiled the isoflavone content in dry seeds from 211 soybean [Glycine max (L.) Merr.] accessions grown across five environments. Broad and discernible phenotypic variations were observed among accessions, regions, and years of growth. Twenty-six single-nucleotide polymorphisms (SNPs) associated with the sum of glycitein (GLE), glycitin (GL), 6″-O-acetylglycitin (AGL), and 6″-O-malonylglycitin (MGL) contents were detected in multiple environments via a genome-wide association study (GWAS). These SNPs were located on chromosome 11 (8,148,438 bp to 8,296,956 bp, renamed qGly11-01). Glyma.11g108300 (GmGLY1), a gene that encodes a P450 family protein, was identified via sequence variation analysis, functional annotation, weighted gene coexpression network analysis (WGCNA), and expression profile analysis of candidate gene, and hairy roots transformation in soybean. Overexpression of GmGLY1 increased the glycitein content (GLC) in soybean hairy roots and transgenic seeds, while CRISPR/Cas9-generated mutants exhibited decreased GLC and increased daidzein content (DAC). Haplotype analysis revealed that GmGLY1 allelic variations significantly affect the GLC accumulation. These findings enhance our understanding of genes influencing GLC in soybean and may guide breeding for lines with high and stable GLC.

Ferroptosis contributes to airway epithelial E-cadherin disruption in a mixed granulocytic asthma mouse model.

Aberrant expression of airway epithelial E-cadherin is a key feature of asthma, yet the underlying mechanisms are largely unknown. Ferroptosis is a novel form of regulated cell death involved in asthma pathogenesis. This study was aimed to evaluate the role of ferroptosis and to investigate whether ferroptosis mediates E-cadherin disruption in mixed granulocyte asthma (MGA). Two murine models of MGA were established using toluene diisocyanate (TDI) or ovalbumin with Complete Freund's Adjuvant (OVA/CFA). Specific antagonists of ferroptosis, including Liproxstatin-1 (Lip-1) and Ferrostatin-1 (Fer-1) were given to the mice. The allergen-exposed mice displayed markedly shrunk mitochondria in the airway epithelia, with decreased volume and denser staining accompanied by down-regulated GPX4 as well as up-regulated FTH1 and malondialdehyde, which are markers of ferroptosis. Decreased pulmonary expression of E-cadherin was also observed, with profound loss of membrane E-cadherin in the airway epithelia, as well as increased secretion of sE-cadherin. Treatment with Lip-1 not only showed potent protective effects against the allergen-induced airway hyperresponsiveness and inflammatory responses, but also rescued airway epithelial E-cadherin expression and inhibited the release of sE-cadherin. Taken together, our data demonstrated that ferroptosis mediates airway epithelial E-cadherin dysfunction in MGA.

Ferroptosis mediates airway epithelial E-cadherin dysfunction in LPS-induced acute lung injury.

BACKGROUND: Loss of E-cadherin in the airway epithelial cells is a critical contributor to the development of ALI/ARDS. Yet the underlying mechanisms are largely unknown. Increasing evidences have revealed the significance of ferroptosis in the pathophysiological process of ALI/ARDS. The aim of this study was to investigate the role of ferroptosis in dysregulation of airway epithelial E-cadherin in ALI/ARDS. METHODS: BALB/c mice were subjected to intratracheal instillation of lipopolysaccharide (LPS) to establish an ALI model. Two inhibitors of ferroptosis, liproxstatin-1 (Lip-1, at the dose of 10 mg/kg and 30 mg/kg) and ferrostatin-1 (Fer-1, at the dose of 1 mg/kg and 5 mg/kg), were respectively given to the mice through intraperitoneal injection after LPS challenge. The expression of ferroptotic markers, full-length E-cadherin and soluble E-cadherin (sE-cadherin) were both detected. RESULTS: LPS exposure dramatically down-regulated pulmonary expression of E-cadherin in mice, with profound loss of membrane E-cadherin in the airway epithelial cells and increased secretion of sE-cadherin in the airway lumen. At the same time, we found that the mitochondrial of airway epithelial cells in LPS-exposed mice exhibited significant morphological alterations that are hallmark features of ferroptosis, with smaller volume and increased membrane density. Other makers of ferroptosis were also detected, including increased cytoplasmic levels of iron and lipid peroxidates (MDA), as well as decreased GPX4 expression. 30 mg/kg of Lip-1 not only showed potent protective effects against the LPS-induced injury, inflammation, edema of the lung in those mice, but also rescued airway epithelial E-cadherin expression and decreased the release of sE-cadherin through inhibiting ferroptosis. While no noticeable changes induced by LPS were observed in mice treated with Lip-1 at 10 mg/kg nor Fer-1 at 1 mg/kg or 5 mg/kg. CONCLUSIONS: Taken together, these data demonstrated that ferroptosis mediates airway epithelial E-cadherin dysfunction in LPS-induced ALI.

GLUT1 contributes to impaired epithelial tight junction in the late phase of acute lung injury.

Dysfunction of epithelial barrier is crucial for the development of acute lung injury (ALI). This study was aimed to evaluate the role of glucose transporter 1 (GLUT1) in dysregulation of epithelial tight junction in ALI. GLUT1 was inhibited with specific antagonists WZB117 or BAY876 to see the effects on epithelial tight junction in a well-established LPS-induced mouse ALI model as well as in vitro cultured epithelial cells. Pharmacological inhibition of GLUT1 with WZB117 at either a low or high dose had no effects on lung injury and inflammation 24 h after LPS challenge, but significantly decreased the pulmonary inflammatory responses induced by LPS at 72 h with a high dose, which was verified by treatment with BAY876. WZB117 or BAY876 also recovered the expression of epithelial tight junction proteins ZO-1 and occludin. In cultured BEAS-2B and A549 cells, LPS induced increased GLUT1 expression, accompanied by decreased expression of tight junction protein ZO-1 and occludin. Blockade of GLUT1 restored LPS-induced disruption of ZO-1 and occludin in BEAS-2B rather than A549. Taken together, our results showed that GLUT1 is responsible for dysfunction of epithelial tight junctions in the late phase of LPS-induced ALI.

Multiplex CRISPR/Cas9-mediated metabolic engineering increases soya bean isoflavone content and resistance to soya bean mosaic virus.

Isoflavonoids, which include a variety of secondary metabolites, are derived from the phenylpropanoid pathway and are distributed predominantly in leguminous plants. These compounds play a critical role in plant-environment interactions and are beneficial to human health. Isoflavone synthase (IFS) is a key enzyme in isoflavonoid synthesis and shares a common substrate with flavanone-3-hydroxylase (F3H) and flavone synthase II (FNS II). In this study, CRISPR/Cas9-mediated multiplex gene-editing technology was employed to simultaneously target GmF3H1, GmF3H2 and GmFNSII-1 in soya bean hairy roots and plants. Various mutation types and frequencies were observed in hairy roots. Higher mutation efficiencies were found in the T0 transgenic plants, with a triple gene mutation efficiency of 44.44%, and these results of targeted mutagenesis were stably inherited in the progeny. Metabolomic analysis of T0 triple-mutants leaves revealed significant improvement in isoflavone content. Compared with the wild type, the T3 generation homozygous triple mutants had approximately twice the leaf isoflavone content, and the soya bean mosaic virus (SMV) coat protein content was significantly reduced by one-third after infection with strain SC7, suggesting that increased isoflavone content enhanced the leaf resistance to SMV. The isoflavone content in the seeds of T2 triple mutants was also significantly increased. This study provides not only materials for the improvement of soya bean isoflavone content and resistance to SMV but also a simple system to generate multiplex mutations in soya bean, which may be beneficial for further breeding and metabolic engineering.