The ß3/5 Integrin-MMP9 Axis Regulates Pulmonary Inflammatory Response and Endothelial Leakage in Acute Lung Injury.

BACKGROUND: Acute lung injury (ALI) is a severe respiratory disease with high rates of morbidity and mortality. Many mediators regarding endogenous or exogenous are involved in the pathophysiology of ALI. Here, we have uncovered the involvement of integrins and matrix metalloproteinases, as critical determinants of excessive inflammation and endothelial permeability, in the regulation of ALI. METHODS: Inflammatory cytokines were measured by quantitative real-time PCR for mRNA levels and ELISA for secretion levels. Endothelial permeability assay was detected by the passage of rhodamine B isothiocyanate-dextran. Mice lung permeability was assayed by Evans blue albumin (EBA). Western blot was used for protein level measurements. The intracellular reactive oxygen species (ROS) were evaluated using a cell-permeable probe, DCFH-DA. Intratracheal injection of lipopolysaccharide (LPS) into mice was conducted to establish the lung injury model. RESULTS: Exogenous MMP-9 significantly aggravated the inflammatory response and permeability in mouse pulmonary microvascular endothelial cells (PMVECs) treated by LPS, whereas knockdown of MMP-9 exhibited the opposite phenotypes. Knockdown of integrin ß3 or ß5 in LPS-treated PMVECs significantly downregulated MMP-9 expression and decreased inflammatory response and permeability in the presence or absence of exogenous MMP-9. Additionally, the interaction of MMP-9 and integrin ß5 was impaired by a ROS scavenger, which further decreased the pro-inflammatory cytokines production and endothelial leakage in PMVECs subjected to co-treatment (LPS with exogenous MMP-9). In vivo studies, exogenous MMP-9 treatment or knockdown ß3 integrin significantly decreased survival in ALI mice. Notably, knockdown of ß5 integrin alone had no remarkable effect on survival, but which combined with anti-MMP-9 treatment significantly improved the survival by ameliorating excessive lung inflammation and permeability in ALI mice. CONCLUSION: These findings support the ß3/5 integrin-MMP-9 axis as an endogenous signal that could play a pivotal role in regulating inflammatory response and alveolar-capillary permeability in ALI.

Development and psychometric validity of the perioperative anxiety scale-7 (PAS-7).

BACKGROUND: Preoperative anxiety is a common psychological reaction in perioperative patients. The absence of a valid measurement tool hinders the evaluation of interventions to treat preoperative anxiety in China. This study aims to develop the Perioperative Anxiety Scale-7 (PAS-7) and test its reliability, validity, and cut-off value. METHODS: A total of 280 patients over 16 years old (M = 55.1, SD = 14.3) who were undergoing elective surgery were recruited to complete the PAS-7 and the Generalized Anxiety Disorder-7 scale (GAD-7) one day before surgery. RESULTS: The PAS-7 included seven items divided into two dimensions: mental anxiety and somatic anxiety. These two dimensions could explain 74.294% of the population variance. The internal consistency of each dimension ranged from 0.761-0.933. The confirmatory factor analysis showed that the model fit of the scale was good (χ2= 34.798, df = 13, χ2/df = 2.677, CFI = 0.949, TLI = 0.918, SRMR = 0.057, RMSEA = 0.115). The correlations between the GAD-7 and each dimension and the scale's total score were significant (0.711-0.789). A cut-off score of 8, maximizing the Youden Index, yielded a sensitivity of 75% and a specificity of 84.6% (95% CI: 0.88 ~ 0.97). CONCLUSIONS: The PAS-7 had good reliability and validity and could be used as an effective tool to evaluate preoperative anxiety.

Transcriptome Sequencing Revealed an Inhibitory Mechanism of Aspergillus flavus Asexual Development and Aflatoxin Metabolism by Soy-Fermenting Non-Aflatoxigenic Aspergillus.

Aflatoxins (AFs) have always been regarded as the most effective carcinogens, posing a great threat to agriculture, food safety, and human health. Aspergillus flavus is the major producer of aflatoxin contamination in crops. The prevention and control of A. flavus and aflatoxin continues to be a global problem. In this study, we demonstrated that the cell-free culture filtrate of Aspergillus oryzae and a non-aflatoxigenic A. flavus can effectively inhibit the production of AFB1 and the growth and reproduction of A. flavus, indicating that both of the non-aflatoxigenic Aspergillus strains secrete inhibitory compounds. Further transcriptome sequencing was performed to analyze the inhibitory mechanism of A. flavus treated with fermenting cultures, and the results revealed that genes involved in the AF biosynthesis pathway and other biosynthetic gene clusters were significantly downregulated, which might be caused by the reduced expression of specific regulators, such as AflS, FarB, and MtfA. The WGCNA results further revealed that genes involved in the TCA cycle and glycolysis were potentially involved in aflatoxin biosynthesis. Our comparative transcriptomics also revealed that two conidia transcriptional factors, brlA and abaA, were found to be significantly downregulated, which might lead to the downregulation of conidiation-specific genes, such as the conidial hydrophobins genes rodA and rodB. In summary, our research provides new insights for the molecular mechanism of controlling AF synthesis to control the proliferation of A. flavus and AF pollution.

G Protein α Subunit GpaB is Required for Asexual Development, Aflatoxin Biosynthesis and Pathogenicity by Regulating cAMP Signaling in Aspergillus flavus.

The heterotrimeric G proteins are critical for signal transduction and function in numerous biological processes including vegetative growth, asexual development and fungal virulence in fungi. Here, we identified four G protein alpha subunits (GanA, GpaB, FadA and GaoC) in the notorious Aflatoxin-producing fungus Aspergillus flavus. GanA, GpaB and FadA have homologues in other fungal species, while GaoC is a novel one. Here, we showed that the loss function of gpaB displayed a defect in conidiophore formation and considerably reduced expression levels of conidia-specific genes brlA and abaA. A decreased viability of cell wall integrity stress and oxidative stress were also found in the ∆gpaB mutant. More importantly, aflatoxin (AF) biosynthesis and infection on crop seeds were severely impaired in the gpaB-deficient mutant. Further analyses demonstrated that the intracellular cAMP levels significantly reduced in the gpaB-deficient mutant compared to wildtype strains. Additionally, an alteration of PKA activities in the ∆gpaB mutant was also found. Overall, our results indicated that GpaB played diverse roles in asexual sporulation, AF biosynthesis and virulence by regulating cAMP signaling in Aspergillus flavus.

Simvastatin inhibits inflammatory response in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages through the microRNA-22/Cyr61 axis.

Simvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has been shown to improve atherosclerosis (AS) via its anti-inflammatory activity. Recently, several studies have reported the involvement of macrophages in chronic inflammation associated with AS. However, it is unknown whether macrophages participate in the anti-inflammatory activity of simvastatin in AS. This study was designed to investigate the roles and underlying mechanisms of simvastatin in LPS-stimulated RAW264.7 macrophages. First, we examined the anti-inflammatory effects of simvastatin on LPS-treated macrophage RAW264.7 cells using an enzyme-linked immunosorbent assay (ELISA) and a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Then, a microarray assay was used to analyze the microRNA (miRNA) expression profile in RAW264.7 cells incubated with or without simvastatin in the presence of LPS. MicroRNA-22 (miR-22) with the highest change was validated independently by qRT-PCR. Luciferase reporter assays were conducted to determine the association between miR-22 and the cysteine-rich protein 61 (Cyr61). Subsequently, we investigated the molecular mechanism by which miR-22 functions in the anti-inflammation of simvastatin in LPS-stimulated macrophages. We found that simvastatin treatment could significantly inhibit inflammation by modulating the expression of mediators, such as IL-1ß, TNF-α and IL-6, whose expression were increased remarkably in the activated RAW264.7 cells. miR-22 was found to be one of the most significantly upregulated miRNAs in LPS-stimulated RAW264.7 macrophages after treatment with simvastatin. Pre-treatment of simvastatin in LPS-stimulated RAW264.7 macrophages enhanced miR-22 expression in a dose dependent manner. Interestingly, Cyr61, a novel pro-inflammatory factor involved in the pathogenesis of atherosclerosis (AS), was identified as a direct target of miR-22. Overexpression of miR-22 enhanced the anti-inflammatory effects of simvastatin, whereas inhibition of miR-22 had an opposite effect. More importantly, further study demonstrated that the knockdown of Cyr61 by siRNA could attenuate the inhibitory effects of miR-22 inhibition on anti-inflammatory activities of simvastatin. The results clearly show that simvastatin inhibits the inflammation response in LPS-stimulated RAW264.7 macrophages through the miR-22/Cyr61 axis and suggests that targeting the miR-22/Cyr61 axis may be a promising molecular target for AS therapy.