The Asthma Risk Gene, GSDMB, Promotes Mitochondrial DNA-induced ISGs Expression.

Released mitochondrial DNA (mtDNA) in cells activates cGAS-STING pathway, which induces expression of interferon-stimulated genes (ISGs) and thereby promotes inflammation, as frequently seen in asthmatic airways. However, whether the genetic determinant, Gasdermin B (GSDMB), the most replicated asthma risk gene, regulates this pathway remains unknown. We set out to determine whether and how GSDMB regulates mtDNA-activated cGAS-STING pathway and subsequent ISGs induction in human airway epithelial cells. Using qPCR, ELISA, native polyacrylamide gel electrophoresis, co-immunoprecipitation and immunofluorescence assays, we evaluated the regulation of GSDMB on cGAS-STING pathway in both BEAS-2B cells and primary normal human bronchial epithelial cells (nHBEs). mtDNA was extracted in plasma samples from human asthmatics and the correlation between mtDNA levels and eosinophil counts was analyzed. GSDMB is significantly associated with RANTES expression in asthmatic nasal epithelial brushing samples from the Genes-environments and Admixture in Latino Americans (GALA) II study. Over-expression of GSDMB promotes DNA-induced IFN and ISGs expression in bronchial epithelial BEAS-2B cells and nHBEs. Conversely, knockout of GSDMB led to weakened induction of interferon (IFNs) and ISGs in BEAS-2B cells. Mechanistically, GSDMB interacts with the C-terminus of STING, promoting the translocation of STING to Golgi, leading to the phosphorylation of IRF3 and induction of IFNs and ISGs. mtDNA copy number in serum from asthmatics was significantly correlated with blood eosinophil counts especially in male subjects. GSDMB promotes the activation of mtDNA and poly (dA:dT)-induced activation of cGAS-STING pathway in airway epithelial cells, leading to enhanced induction of ISGs.

Gasdermin B, an asthma-susceptibility gene, promotes MAVS-TBK1 signalling and airway inflammation.

RATIONALE: Respiratory virus-induced inflammation is the leading cause of asthma exacerbation, frequently accompanied by induction of interferon-stimulated genes (ISGs). How asthma-susceptibility genes modulate cellular response upon viral infection by fine-tuning ISG induction and subsequent airway inflammation in genetically susceptible asthma patients remains largely unknown. OBJECTIVES: To decipher the functions of gasdermin B (encoded by GSDMB) in respiratory virus-induced lung inflammation. METHODS: In two independent cohorts, we analysed expression correlation between GSDMB and ISG s. In human bronchial epithelial cell line or primary bronchial epithelial cells, we generated GSDMB-overexpressing and GSDMB-deficient cells. A series of quantitative PCR, ELISA and co-immunoprecipitation assays were performed to determine the function and mechanism of GSDMB for ISG induction. We also generated a novel transgenic mouse line with inducible expression of human unique GSDMB gene in airway epithelial cells and infected the mice with respiratory syncytial virus to determine the role of GSDMB in respiratory syncytial virus-induced lung inflammation in vivo. RESULTS: GSDMB is one of the most significant asthma-susceptibility genes at 17q21 and acts as a novel RNA sensor, promoting mitochondrial antiviral-signalling protein (MAVS)-TANK binding kinase 1 (TBK1) signalling and subsequent inflammation. In airway epithelium, GSDMB is induced by respiratory viral infections. Expression of GSDMB and ISGs significantly correlated in respiratory epithelium from two independent asthma cohorts. Notably, inducible expression of human GSDMB in mouse airway epithelium led to enhanced ISGs induction and increased airway inflammation with mucus hypersecretion upon respiratory syncytial virus infection. CONCLUSIONS: GSDMB promotes ISGs expression and airway inflammation upon respiratory virus infection, thereby conferring asthma risk in risk allele carriers.

Identification of a distal enhancer regulating hedgehog interacting protein gene in human lung epithelial cells.

BACKGROUND: An intergenic region at chromosome 4q31 is one of the most significant regions associated with COPD susceptibility and lung function in GWAS. In this region, the implicated causal gene HHIP has a unique epithelial expression pattern in adult human lungs, in contrast to dominant expression in fibroblasts in murine lungs. However, the mechanism underlying the species-dependent cell type-specific regulation of HHIP remains largely unknown. METHODS: We employed snATAC-seq analysis to identify open chromatin regions within the COPD GWAS region in various human lung cell types. ChIP-quantitative PCR, reporter assays, chromatin conformation capture assays and Hi-C assays were conducted to characterize the regulatory element in this region. CRISPR/Cas9-editing was performed in BEAS-2B cells to generate single colonies with stable knockout of the regulatory element. RT-PCR and Western blot assays were used to evaluate expression of HHIP and epithelial-mesenchymal transition (EMT)-related marker genes. FINDINGS: We identified a distal enhancer within the COPD 4q31 GWAS locus that regulates HHIP transcription at baseline and after TGFß treatment in a SMAD3-dependent, but Hedgehog-independent manner in human bronchial epithelial cells. The distal enhancer also maintains chromatin topological domains near 4q31 locus and HHIP gene. Reduced HHIP expression led to increased EMT induced by TGFß in human bronchial epithelial cells. INTERPRETATION: A distal enhancer regulates HHIP expression both under homeostatic condition and upon TGFß treatment in human bronchial epithelial cells. The interaction between HHIP and TGFß signalling possibly contributes to COPD pathogenesis. FUNDING: Supported by NIH grants R01HL127200, R01HL148667 and R01HL162783 (to X. Z).

Microstructure and Properties of CoCrNi/Nano-TiC/Micro-TiB2 Composite Coatings Prepared via Laser Cladding.

Laser cladding was used to prepare CoCrNi-xTiC-xTiB2 (x = 0, 5, 15 wt.%) composite coatings on 316L stainless steel. Then, ceramic mass fraction effects on the microstructure and properties were investigated. Results show viable metallurgical bonding between the coating and the substrate, with no apparent pores or cracks. The addition of ceramics transformed the coating phase from a single-phase face-centered cubic (FCC) to a multi-phase FCC+TiC+TiB2. TiC and TiB2 increased the hardness of the CoCrNi-xTiC-xTiB2 coating from 209.71 HV to 494.77 HV by grain refinement and diffusion strengthening. The substrate wear loss was 0.0088 g, whereas the CoCrNi-xTiC-xTiB2 (x = 15%) coating wear loss was only 0.0012 g. Moreover, the overall wear mechanism of the coating was changed: the substrate wear mechanism was used for abrasive wear, adhesive wear and fatigue wear, and the coating with the addition of 15 wt.% nano-TiC and 15 wt.% micro-TiB2 was the wear mechanism for pitting fatigue wear.

The Effects of Ethanol and Rutin on the Structure and Gel Properties of Whey Protein Isolate and Related Mechanisms.

The effects of different levels of rutin (0, 0.05%, 0.1%, 0.2% and 0.3% w/v) and ethanol on the structure and gel properties of whey protein isolate (WPI) were examined. The results showed that the addition of ethanol promoted the gel formation of WPI. The addition of rutin increased the gel strength of WPI and maintained the water-holding capacity of the gel. Ethanol caused an increase in thiol content and surface hydrophobicity, but rutin decreased the thiol content and surface hydrophobicity of WPI. The particle size, viscosity and viscoelasticity of WPI increased at rutin levels of 0.2% and 0.3%, indicating that rutin caused cross-linking and aggregation of WPI, but rutin had no significant effect on the zeta-potential, indicating that electrostatic interactions were not the main force causing the changes in protein conformation and gel properties. Ethanol and rutin improved the gel properties of WPI possibly by inducing cross-linking of WPIs via hydrophobic and covalent interactions.

Ambient Air Pollution and Adverse Pregnancy Outcomes in Wuhan, China.

Background: Several recent studies have suggested that maternal exposures to air pollution and temperature extremes might contribute to low birth weight (LBW), preterm birth (PTB), and other outcomes that can adversely affect infant health. At the time the current study began, most other studies had been conducted in the United States or Europe. Dr. Zhengmin Qian proposed to extend work he had done on ambient particulate air pollution and daily mortality in Wuhan, China (Qian et al. 2010), as part of the HEIsponsored Public Health and Air Pollution in Asia program, to study adverse birth outcomes. Wuhan is the capital city of Hubei province, has a large population of about 6.4 million within the urban study area, experiences temperature extremes, and generally has higher air pollution levels than those observed in the United States and Europe, thus providing a good opportunity to explore questions about air pollution and health. Approach: Qian and colleagues planned a cohort and nested case­control design with four specific aims, examining whether increased exposures to air pollutants (PM2.5, PM10, SO2, NO2, O3, and CO) during vulnerable pregnancy periods were associated with increased rates of PTB, LBW (<2500 g), or intrauterine growth retardation (IUGR, defined as having a birth weight below the 10th percentile of singleton live births in Wuhan) after adjusting for major risk factors and whether the associations were confounded by copollutant exposures, affected by residual confounding, or modified by temperature extremes, socioeconomic status (SES), or secondhand smoke (SHS) exposure. The cohort study included 95,911 births that occurred from June 10, 2011, to June 9, 2013, and met typical prespecified inclusion criteria used in other birth outcome studies. The case­control study included 3146 cases (PTB, LBW, or both, but not IUGR) and 4263 controls (matched to the cases by birth month) for whom investigators were able to complete home visits and questionnaires. The investigators obtained air pollution and daily weather data for August 2010 to June 2013 from nine monitoring stations representing background air pollution sites in seven Wuhan inner-city districts. Only two of these stations provided PM2.5 data. For the cohort study, the investigators assigned exposures to mothers according to the daily mean concentrations from the monitor nearest the residential community in which the mother lived at the time of the birth. For the case­control study, they assigned exposures based on the inverse distance weighted average of daily mean concentrations from the three nearest monitors, for all but PM2.5 for which the method was not specified. They also collected data on various factors that might confound or modify the impact of the pollutants on the adverse outcomes, including data collected in the cohort from mothers at the time of delivery and, in the case­control study, from questionnaires administered to mothers. In the case­control study, covariates representing SES (as indicated by the mother's educational attainment and household income) and SHS exposures were of particular interest. The primary statistical analyses of the pollutant associations with PTB, LBW, and IUGR were conducted using logistic regression models. In the cohort study, exposures during the pregnancy period of interest (full term, trimesters, and selected months) were included as continuous variables. In the case­control study, the exposures were modeled as binary variables (i.e., above or below the median pollutant concentrations). Numerous sensitivity analyses were conducted. Results and interpretation: Although originally planning a nested case­control study, the investigators encountered challenges that led them to analyze the cohort and case­control studies using different ways of assigning exposures and characterizing them in their statistical models. These decisions precluded direct comparisons between the sets of results, making it difficult to answer the questions about residual confounding that nested case­control studies are designed to answer. The odds ratios from the two study designs using different exposures also have different interpretations. Still, one can ask whether the sets of findings were qualitatively consistent with each other or with those of similar studies. There were some similarities. Both studies suggested that increased PM(2.5), PM(10), CO, and O(3) exposures over the full pregnancy were associated with small increases in the odds of PTB (the case­control study also showed an association with NO2) and that increased PM(2.5) exposures were associated with significantly increased odds of LBW. However, most of the other pollutants had no effect on LBW, except CO in the cohort study and O(3) in the case­control study, both of which increased the odds of LBW. The exposures over the entire pregnancy were generally associated with decreased odds of IUGR. Adjustments for potential confounders were greatest for the delivery covariates. The investigators found no systematic association of any of these outcomes with particular trimesters or months, another result that differed from those of some other studies. They found little evidence that their main results were confounded or modified by the presence of copollutants, although with the exception of O3, most of the pollutants were highly correlated, making it difficult to disentangle the effects of individual pollutants. Could the two sets of data be analyzed in a more comparable way, as in a standard nested case­control study? At the Committee's request, the investigators reanalyzed the case­control data using the same exposures and models as in the cohort study. The results were strikingly different from those using the inverse distance weighted exposures, modeled as binary variables ­ the pollutants had either no effect or an apparent beneficial effect on PTB and LBW. The Committee was not convinced by the explanations offered for these differences, leaving the reasons for them unresolved. Conclusions: This study set out to answer important questions about the effects of air pollution exposure on three measures of adverse birth outcomes ­ LBW, PTB, and IUGR ­ in a large cohort of mothers and newborns in Wuhan, China. Given the cohort size, high pollution levels and temperatures, and detailed covariate data, the investigators were well poised to address these questions. They sought to pattern their work on other studies of birth outcomes, were very responsive to Committee questions, and provided many additional analyses and explanations. In the Committee's view, however, the study was unable to address with confidence several of its specific aims. Most important, the differences in results when the case­control data were analyzed with different exposure metrics remain unexplained, raising concerns about the ability to draw conclusions from subsequent analyses assessing residual confounding and effect modification by temperature extremes, SES, and SHS exposure. Consequently, any individual findings from the cohort and case­control studies should be considered suggestive rather than conclusive, and should be interpreted carefully together.

Pre-Pregnancy Body Mass Index, Gestational Weight Gain, and Birth Weight: A Cohort Study in China.

OBJECTIVE: To assess whether pre-pregnancy body mass index (BMI) modify the relationship between gestational weight gain (GWG) and child birth weight (specifically, presence or absence of low birth weight (LBW) or presence of absence of macrosomia), and estimates of the relative risk of macrosomia and LBW based on pre-pregnancy BMI were controlled in Wuhan, China. METHODS: From June 30, 2011 to June 30, 2013. All data was collected and available from the perinatal health care system. Logistic regression models were used to estimate the independent association among pregnancy weight gain, LBW, normal birth weight, and macrosomia within different pre-pregnancy BMI groups. We built different logistic models for the 2009 Institute of Medicine (IOM) Guidelines and Chinese-recommended GWG which was made from this sample. The Chinese-recommended GWG was derived from the quartile values (25th-75th percentiles) of weight gain at the time of delivery in the subjects which comprised our sample. RESULTS: For LBW children, using the recommended weight gain of the IOM and Chinese women as a reference, the OR for a pregnancy weight gain below recommendations resulted in a positive relationship for lean and normal weight women, but not for overweight and obese women. For macrosomia, considering the IOM's recommended weight gain as a reference, the OR magnitude for pregnancy weight gain above recommendations resulted in a positive correlation for all women. The OR for a pregnancy weight gain below recommendations resulted in a negative relationship for normal BMI and lean women, but not for overweight and obese women based on the IOM recommendations, significant based on the recommended pregnancy weight gain for Chinese women. Of normal weight children, 56.6% were above the GWG based on IOM recommendations, but 26.97% of normal weight children were above the GWG based on Chinese recommendations. CONCLUSIONS: A GWG above IOM recommendations might not be helpful for Chinese women. We need unified criteria to classify adult BMI and to expand the sample size to improve representation and to elucidate the relationship between GWG and related outcomes for developing a Chinese GWG recommendation.

Phosphorylation and Interaction with the 14-3-3 Protein of the Plasma Membrane H+-ATPase are Involved in the Regulation of Magnesium-Mediated Increases in Aluminum-Induced Citrate Exudation in Broad Bean (Vicia faba. L).

Several studies have shown that external application of micromolar magnesium (Mg) can increase the resistance of legumes to aluminum (Al) stress by enhancing Al-induced citrate exudation. However, the exact mechanism underlying this regulation remains unknown. In this study, the physiological and molecular mechanisms by which Mg enhances Al-induced citrate exudation to alleviate Al toxicity were investigated in broad bean. Micromolar concentrations of Mg that alleviated Al toxicity paralleled the stimulation of Al-induced citrate exudation and increased the activity of the plasma membrane (PM) H(+)-ATPase. Northern blot analysis shows that a putative MATE-like gene (multidrug and toxic compound extrusion) was induced after treatment with Al for 4, 8 and 12 h, whereas the mRNA abundance of the MATE-like gene showed no significant difference between Al plus Mg and Al-only treatments during the entire treatment period. Real-time reverse transcription-PCR (RT-PCR) and Western blot analyses suggest that the transcription and translation of the PM H(+)-ATPase were induced by Al but not by Mg. In contrast, immunoprecipitation suggests that Mg enhanced the phosphorylation levels of VHA2 and its interaction with the vf14-3-3b protein under Al stress. Taken together, our results suggest that micromolar concentrations of Mg can alleviate the Al rhizotoxicity by increasing PM H(+)-ATPase activity and Al-induced citrate exudation in YD roots. This enhancement is likely to be attributable to Al-induced increases in the expression of the MATE-like gene and vha2 and Mg-induced changes in the phosphorylation levels of VHA2, thus changing its interaction with the vf14-3-3b protein.

Displacement-based back-analysis of the model parameters of the Nuozhadu high earth-rockfill dam.

The parameters of the constitutive model, the creep model, and the wetting model of materials of the Nuozhadu high earth-rockfill dam were back-analyzed together based on field monitoring displacement data by employing an intelligent back-analysis method. In this method, an artificial neural network is used as a substitute for time-consuming finite element analysis, and an evolutionary algorithm is applied for both network training and parameter optimization. To avoid simultaneous back-analysis of many parameters, the model parameters of the three main dam materials are decoupled and back-analyzed separately in a particular order. Displacement back-analyses were performed at different stages of the construction period, with and without considering the creep and wetting deformations. Good agreement between the numerical results and the monitoring data was obtained for most observation points, which implies that the back-analysis method and decoupling method are effective for solving complex problems with multiple models and parameters. The comparison of calculation results based on different sets of back-analyzed model parameters indicates the necessity of taking the effects of creep and wetting into consideration in the numerical analyses of high earth-rockfill dams. With the resulting model parameters, the stress and deformation distributions at completion are predicted and analyzed.