Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

Mutations of cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial ligand-gated anion channel, are associated with the lethal genetic disease cystic fibrosis. The CFTR G551D mutation impairs ATP hydrolysis and thereby makes CFTR refractory to cAMP stimulation. Both wild-type (WT) and G551D CFTR have been implicated in regulatory volume decrease (RVD), but the underlying mechanism remains incompletely understood. Here, we show that the channel activity of both WT and G551D CFTR is directly stimulated by mechanical perturbation induced by cell swelling at the single-channel, cellular, and tissue levels. Hypotonicity activated CFTR single channels in cell-attached membrane patches and WT-CFTR-mediated short-circuit current (Isc) in Calu-3 cells, and this was independent of Ca(2+)and cAMP/PKA signaling. Genetic suppression and ablation but not G551D mutation of CFTR suppressed the hypotonicity- and stretch-inducedIscin Calu-3 cells and mouse duodena. Moreover, ablation but not G551D mutation of the CFTR gene inhibited the RVD of crypts isolated from mouse intestine; more importantly, CFTR-specific blockers markedly suppressed RVD in both WT- and G551D CFTR mice, demonstrating for the first time that the channel activity of both WT and G551D CFTR is required for epithelial RVD. Our findings uncover a previously unrecognized mechanism underlying CFTR involvement in epithelial RVD and suggest that the mechanosensitivity of G551D CFTR might underlie the mild phenotypes resulting from this mutation.-Xie, C., Cao, X., Chen, X, Wang, D., Zhang, W. K., Sun, Y., Hu, W., Zhou, Z., Wang, Y., Huang, P. Mechanosensitivity of wild-type and G551D cystic fibrosis transmembrane conductance regulator (CFTR) controls regulatory volume decrease in simple epithelia.

Keratin K18 increases cystic fibrosis transmembrane conductance regulator (CFTR) surface expression by binding to its C-terminal hydrophobic patch.

BACKGROUND: CFTR function is tightly regulated by many interacting proteins. RESULTS: Intermediate filament protein keratin 18 increases the cell surface expression of CFTR by interacting with the C-terminal hydrophobic patch of CFTR. CONCLUSION: K18 controls the function of CFTR. SIGNIFICANCE: These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis. Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR-binding protein by various approaches. We mapped a highly conserved "hydrophobic patch" ((1413)FLVI(1416)) in the CFTR C-terminus, known to determine plasmalemmal CFTR stability, as the K18-binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR biosynthesis, maturation, or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18(-/-) mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with the CFTR C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis.

[Effects of reducing chemical fertilizers combined with organic fertilizers on soil microbial community in litchi orchards]. / å‡æ–½åŒ–è‚¥é æ–½æœ‰æœºè‚¥å¯¹è”æžå›­åœŸå£¤å¾®ç”Ÿç‰©åŒºç³»çš„å½±å“.

Organic fertilizer application can replace a part of chemical fertilizer (CF) to improve the quality and efficiency of litchi production. To further explore the soil microbiological mechanism, with 19-year-old 'Feizixiao' litchi trees as the research objects, we examined the effects of two consecutive years of reduced CF applications (average 21.5% of total nutrients) combined with sheep manure (OF) and bio-organic fertilizers (BIO) on soil microbial diversity, community composition and differential microorganisms. The results showed that reducing the application of chemical fertilizers and combining it with the application of sheep manure and bio-organic fertilizer for two consecutive years could significantly improve yield and quality. The average increase of yield in the two years was 23.1% and 39.0%, respectively. Soil organic matter content and pH increased significantly in response to the combination treatments. Compared to that in the chemical fertilizer treatment, the contents of soil available phosphorus, potassium, calcium, magnesium, iron, manganese, copper, and zinc displayed an increasing trend in the combination treatments. The application of organic fertilizer increased the diversity of bacteria and fungi in rhizosphere soil, but not in non-rhizosphere soil. Both treatments significantly changed soil microbial community structure, increased eutrophic bacterial groups such as Bacteroides, Proteobacteria, and Bacillus phylum, and reduced anatrophic bacterial groups such as Acidobacteria and Chloroflexus. Compared with CF, the relative abundances of MND1 under OF and TK10, Gemmatimonas, Pseudolabrys, Trichoderma and Botryotrichum under BIO were significantly increased, which was positively correlated with yield. In conclusion, reducing CF and applying organic ferti-lizer for two consecutive years could effectively improve soil pH and nutrient availability, increase rhizosphere microbial richness and diversity, change soil microbial community structure, and shape microbial communities being more conducive to yield and quality improvement.

Organic amendments alleviate early defoliation and increase fruit yield by altering assembly patterns and of microbial communities and enzymatic activities in sandy pear (Pyrus pyrifolia).

Severe early defoliation has become an important factor restricting the development of the pear industry in southern China. However, the assembly patterns of microbial communities and their functional activities in response to the application of bioorganic fertilizer (BIO) or humic acid (HA) in southern China's pear orchards remain poorly understood, particularly the impact on the early defoliation of the trees. We conducted a 3-year field experiment (2017-2019) in an 18-year-old 'Cuiguan' pear orchard. Four fertilization schemes were tested: local custom fertilization as control (CK), CK plus HA (CK-HA), BIO, and BIO plus HA (BIO-HA). Results showed that BIO and BIO-HA application decreased the early defoliation rate by 50-60%, and increased pear yield by 40% compared with the CK and CK-HA treatments. The BIO and BIO-HA application significantly improved soil pH, available nutrient content, total enzyme activity and ecosystem multifunctionality, and also changed the structure of soil bacterial and fungal communities. The genus Acidothermus was positively correlated with the early defoliation rate, while the genus Rhodanobacter was negatively correlated. Additionally, random forest models revealed that the early defoliation rate could be best explained by soil pH, ammonium content, available phosphorus, and total enzyme activity. In conclusion, application of BIO or BIO mixed with HA could have assembled distinct microbial communities and increased total enzyme activity, leading to significant improvement of soil physicochemical traits. The increased availability of soil nutrient thus changed leaf nutrient concentrations and alleviated the early defoliation rate of pear trees in acid red soil in southern China.

The change in microstructure of petioles and peduncles and transporter gene expression by potassium influences the distribution of nutrients and sugars in pear leaves and fruit.

Potassium (K) is one of the most important mineral nutrients required for fruit growth and development and is known as a 'quality element'. To investigate the role of K in more detail, we performed experiments in which seven-year-old pot-grown 'Huangguan' pear trees were treated with three levels of K (0, 0.4, or 0.8 g K2O kg-1 soil). K supply improved the development of vascular bundles in pear petioles and fruit peduncles and enhanced expression of genes involved in nutrients and sugar transport. Different from K and calcium (Ca), magnesium (Mg) concentrations in the leaves, petioles, and fruit peduncles were significantly higher under low K but lower under high K. However, the concentrations of K, Ca, and Mg in fruit all increased as more K was applied. Correspondingly, the expression of leaf Mg transporters (MRS2-1 and MRS2-3) increased under low K, indicating that Mg had an obvious compensation effect on K, while their expression decreased under medium and high K, showing that K had an obvious antagonistic effect on Mg. Except for NIPA2, the expressions of fruit K, Ca, and Mg transporters increased under high K, implying a synergistic effect among them in fruit. The concentration of sorbitol, sucrose, and total sugar in leaves and fruit at maturity significantly increased in response to the supply of K. The increase in sugar concentration was closely related to the up-regulated expression of sucrose transporter (SUT) and sorbitol transporter (SOT) genes. Together, these effects may promote the transport of nutrients and sugar from sources (leaves) to sinks (fruit) and increase the accumulation of sugar in the fruit.

Transcriptome Analysis of Differentially Expressed Genes Induced by Low and High Potassium Levels Provides Insight into Fruit Sugar Metabolism of Pear.

Potassium (K) deficiency is a common abiotic stress that can inhibit the growth of fruit and thus reduce crop yields. Little research has been conducted on pear transcriptional changes under low and high K conditions. Here, we performed an experiment with 7-year-old pot-grown "Huangguan" pear trees treated with low, Control or high K levels (0, 0.4, or 0.8 g·K2O/kg soil, respectively) during fruit enlargement and mature stages. We identified 36,444 transcripts from leaves and fruit using transcriptome sequencing technology. From 105 days after full blooming (DAB) to 129 DAB, the number of differentially expressed genes (DEGs) in leaves and fruit in response to low K increased, while in response to high K, the number of DEGs in leaves and fruit decreased. We selected 17 of these DEGs for qRT-PCR analysis to confirm the RNA sequencing results. Based on GO enrichment and KEGG pathway analysis, we found that low-K treatment significantly reduced K nutrient and carbohydrate metabolism of the leaves and fruit compared with the Control treatment. During the fruit development stages, AKT1 (gene39320) played an important role on K+ transport of the leaves and fruit response to K stress. At maturity, sucrose and acid metabolic pathways were inhibited by low K. The up-regulation of the expression of three SDH and two S6PDH genes involved in sorbitol metabolism was induced by low K, promoting the fructose accumulation. Simultaneously, higher expression was found for genes encoding amylase under low K, promoting the decomposition of the starch and leading the glucose accumulation. High K could enhance leaf photosynthesis, and improve the distribution of the nutrient and carbohydrate from leaf to fruit. Sugar components of the leaves and fruit under low K were regulated by the expression of genes encoding 8 types of hormone signals and reactive oxygen species (ROS). Our data revealed the gene expression patterns of leaves and fruit in response to different K levels during the middle and late stages of fruit development as well as the molecular mechanism of improvement of fruit sugar levels by K and provided a scientific basis for improving fruit quality with supplemental K fertilizers.

Organic electrochemical transistor array for recording transepithelial ion transport of human airway epithelial cells.

An organic electrochemical transistor array is integrated with human airway epithelial cells. This integration provides a novel method to couple transepithelial ion transport with electrical current. Activation and inhibition of transepithelial ion transport are readily detected with excellent time resolution. The organic electrochemical transistor array serves as a promising platform for physiological studies and drug testing.