Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between cAMP pathway and Ca2+signalling. Despite the importance of AC8 in physiology, including cognitive functions and memory, the structural basis of its regulation by G proteins and CaM is not well defined. Here we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to Ca2+/CaM and the stimulatory Gαs protein. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and a small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry, allow us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gβγ, as well as the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.
Background: Gastrointestinal (GI) dysfunction is common in the intensive care unit (ICU), although there is no consensus on biomarkers of GI dysfunction. We aimed to evaluate ultrasound-based gastric antrum measurements and serum intestinal fatty acid-binding protein (IFABP) and citrulline levels in relation to GI dysfunction in critically ill patients.Methods: Adult critically ill patients receiving enteral nutrition and stayed for in the ICU for ≥48 h was included. GI dysfunction was described using Gastrointestinal Dysfunction Score (GIDS). Gastric antrum measurements, including craniocaudal (CC) diameter, anteroposterior diameter, and antral-cross sectional area (CSA), as well as serum levels for IFABP and citrulline, were prospectively recorded at baseline and on day 3 and day 5 of enteral nutrition. The receiver operating characteristic (ROC) analysis was performed to evaluate gastric ultrasound parameters, serum IFABP, and citrulline concentrations in predicting GI dysfunction.Results: Thirty-nine participants with a median age of 60 years were recruited and 46.2% of participants had GI dysfunction. ROC analysis revealed that the cutoff value of CSA score to predict GI dysfunction was 4.48 cm 2 , which provided 72.7% sensitivity and 77.2% specificity (area under the curve = 0.768, 95% CI: 0.555-0.980). At baseline, gastric residual volume was highly correlated with CC diameter and CSA (r = 0.764, P < 0.001 and r = 0.675, P < 0.001, respectively). Serum IFABP and citrulline levels had no correlation with GI dysfunction or gastric ultrasound parameters (P > 0.05).
Conclusion:CSA was associated with GI dysfunction in critically ill patients. Serum IFABP and citrulline concentrations were poor in predicting GI dysfunction.
Although the molecular pathogenesis of hepatocellular carcinoma (HCC) is uncertain, it is known that the epithelial-mesenchymal transition (EMT) mechanism and epigenetic changes have an important role. This study was focused on evaluating the relationship of 3-Deazaneplanocin A (DZNep) with the EMT mechanism, which is a histone methyltransferase inhibitor on HCC and is also known as an enhancer of zeste homolog 2 (EZH2) inhibitor. Cell viability of HepG2 cells (HCC cell line) assessed for DZNep over 72 hours with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Additionally, colony-forming assay, apoptosis assay, RNA isolation, cDNA synthesis, and real-time PCR (RT-PCR) were performed to see the effect of DZNep on HepG2 cells. DZNep reduced cell proliferation for 72 hours, also significantly reduced colony formation in addition it increased the total apoptosis. DZNep on EZH2, E-cadherin, N-cadherin, and Vimentin (Vim) gene expressions was given different results by either decreasing or increasing the expressions. In this study, we observed a positive effect of DZNep on apoptosis and TIMP3 expression level and decreased colony formation. However, it gave complicated results with the level of gene expression E-cadherin and TIMP2, increase the level of Vim and MMP2 expression. Therefore, we think that further studies are necessary to clarify the role of DZNep.
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