ABSTRACT
Methylation patterns in cell-free DNA (cfDNA) have emerged as a promising genomic feature for detecting the presence of cancer and determining its origin. The purpose of this study was to evaluate the diagnostic performance of methylation-sensitive restriction enzyme digestion followed by sequencing (MRE-Seq) using cfDNA, and to investigate the cancer signal origin (CSO) of the cancer using a deep neural network (DNN) analyses for liquid biopsy of colorectal and lung cancer. We developed a selective MRE-Seq method with DNN learning-based prediction model using demethylated-sequence-depth patterns from 63,266 CpG sites using SacII enzyme digestion. A total of 191 patients with stage I-IV cancers (95 lung cancers and 96 colorectal cancers) and 126 noncancer participants were enrolled in this study. Our study showed an area under the receiver operating characteristic curve (AUC) of 0.978 with a sensitivity of 78.1% for colorectal cancer, and an AUC of 0.956 with a sensitivity of 66.3% for lung cancer, both at a specificity of 99.2%. For colorectal cancer, sensitivities for stages I-IV ranged from 76.2 to 83.3% while for lung cancer, sensitivities for stages I-IV ranged from 44.4 to 78.9%, both again at a specificity of 99.2%. The CSO model's true-positive rates were 94.4% and 89.9% for colorectal and lung cancers, respectively. The MRE-Seq was found to be a useful method for detecting global hypomethylation patterns in liquid biopsy samples and accurately diagnosing colorectal and lung cancers, as well as determining CSO of the cancer using DNN analysis.Trial registration: This trial was registered at ClinicalTrials.gov (registration number: NCT04253509) for lung cancer on 5 February 2020, https://clinicaltrials.gov/ct2/show/NCT04253509 . Colorectal cancer samples were retrospectively registered at CRIS (Clinical Research Information Service, registration number: KCT0008037) on 23 December 2022, https://cris.nih.go.kr , https://who.init/ictrp . Healthy control samples were retrospectively registered.
Subject(s)
Cell-Free Nucleic Acids , Colorectal Neoplasms , Lung Neoplasms , Humans , Methylation , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Liquid Biopsy , Gastrointestinal Agents , Colorectal Neoplasms/diagnosis , Colorectal Neoplasms/geneticsABSTRACT
The COVID-19 pandemic is an ongoing global public health threat. COVID-19 is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and binding of the SARS-CoV-2 spike to its receptor, angiotensin-converting enzyme 2 (ACE2), on host cells is critical for viral infection. Here, we developed a luminescent biosensor that readily detects interactions of the spike receptor-binding domain (RBD) and ACE2 in cell culture medium ('SpACE-CCM'), which was based on bimolecular complementation of the split nanoluciferase-fused spike RBD and ectodomain of ACE2 and further engineered to be efficiently secreted from cells by adding a heterologous secretory signal peptide (SSP). Screening of various SSPs identified 'interferon-α+alanine-aspartate' as the SSP that induced the highest activity. The SpACE-CCM biosensor was validated by observing a marked reduction of the activity caused by interaction-defective mutations or in the presence of neutralizing antibodies, recombinant decoy proteins, or peptides. Importantly, the SpACE-CCM biosensor responded well in assay-validating conditions compared with conventional cell lysate-based NanoLuc Binary Technology, indicating its advantage. We further demonstrated the biosensor's versatility by quantitatively detecting neutralizing activity in blood samples from COVID-19 patients and vaccinated individuals, discovering a small molecule interfering with the spike RBD-ACE2 interaction through high-throughput screening, and assessing the cross-reactivity of neutralizing antibodies against SARS-CoV-2 variants. Because the SpACE-CCM is a facile and rapid one-step reaction biosensor that aptly recapitulates the native spike-ACE2 interaction, it would be advantageous in many experimental and clinical applications associated with this interaction.
Subject(s)
Biosensing Techniques , COVID-19 , Humans , SARS-CoV-2 , Angiotensin-Converting Enzyme 2 , Pandemics , Protein Binding , Antibodies, Neutralizing/metabolism , Cell Culture Techniques , Spike Glycoprotein, CoronavirusABSTRACT
The phlorotannin derivative dieckol isolated from Ecklonia cava has been shown to exhibit anti-inflammatory, anti-bacterial, anti-oxidative anti-adipogenic and anti-stenosis activity. However, the role of dieckol in cyclin-dependent kinase 2 (CDK2)/cyclin E signalling, which regulates fibrosis development, has not yet been determined. In this study, we report that dieckol-suppressed cell proliferation through the cell cycle arrest of Hs680.Tr human tracheal fibroblasts. Following consecutive purification, dieckol was identified as a potent bioactive compound. The results showed that dieckol had significant anti-proliferative activity against Hs680.Tr human tracheal fibroblastsWestern blotting analysis also found that dieckol dose-dependently induced the cell cycle arrest of Hs680.Tr fibroblasts in the G0/G1 phase, accompanied by the downregulation of CDK2 and cyclin E and the upregulation of p21 and p53. As attested by molecular docking study, the dieckol interacted with the core interface residues in transforming growth factor-ß receptor with high affinity. These findings suggest that dieckol from E. cava inhibits the cell proliferation of Hs680.Tr, potentially through p21- and p53-mediated G0/G1 cell cycle arrest.
Subject(s)
Benzofurans/pharmacology , Cyclin E , Cyclin-Dependent Kinase 2 , Cyclin-Dependent Kinase Inhibitor p21 , Tumor Suppressor Protein p53 , Cell Cycle , Cell Cycle Checkpoints , Cells, Cultured , Cyclin E/genetics , Cyclin E/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Fibroblasts/metabolism , Humans , Molecular Docking Simulation , Oncogene ProteinsABSTRACT
The objective of this study was to investigate inhibitory effects of a bioactive compound isolated from Ecklonia cava on fibrotic responses to transforming growth factor-ß1 (TGF-ß1)-stimulated Hs680. Tr human tracheal fibroblasts and the associated mechanisms of action. Post consecutive purification, a potent bioactive compound was identified phlorofucofuroeckol A. Phlorofucofuroeckol A significantly suppressed protein expression levels of collagen type I and α-smooth muscle actin (α-SMA) on TGF-ß1-stimulated Hs680. Tr human tracheal fibroblasts. Further mechanistic studies determined that phlorofucofuroeckol A suppressed the phosphorylation of p38, extracellular regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) and SMAD 2/3 in TGF-ß1-stimulated Hs680. Tr human tracheal fibroblasts. Moreover, we could show that phlorofucofuroeckol A inhibits binding of TGF-ß1 to its TGF-ß receptor by molecular docking. Based on the results, we propose that phlorofucofuroeckol A suppresses the MAPKs and SMAD 2/3 pathways and relieves cellular fibrotic activities, thus preventing tracheal fibrosis.
Subject(s)
Benzofurans/pharmacology , Dioxins/pharmacology , Fibroblasts/drug effects , Signal Transduction/drug effects , Trachea/drug effects , Transforming Growth Factor beta1/metabolism , Benzofurans/chemistry , Cell Line , Dioxins/chemistry , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Humans , MAP Kinase Signaling System/drug effects , Phaeophyceae/chemistry , Smad2 Protein/metabolism , Smad3 Protein/metabolism , Trachea/metabolism , Trachea/pathologyABSTRACT
Prolonged endotracheal intubation is the most common cause of tracheal stenosis, which may lead to serious airway obstruction. Development of an endotracheal tube coated with biomaterials that exhibit anti-inflammatory or anti-fibrogenic effects may prevent tracheal stenosis. This study demonstrates that an endotracheal tube coated with phlorotannin, which is present in extracts of the brown alga Ecklonia cava, can prevent tracheal stenosis in a rabbit model. An in vitro study shows that phlorotannin inhibits proliferation of human tracheal fibroblasts treated with transforming growth factor ß1. Phlorotannin-coated endotracheal tubes show steady release of phlorotannin for up to 7 days, and removal of the tube 1 week after insertion reveals a reduction in both fibrogenesis and thickening of tracheal submucosa. Western blot analysis of tracheal tissues after removal of the phlorotannin-coated tube shows decreased protein expression levels of phenotypic markers of fibrosis such as collagen type I and α-smooth muscle actin. The ability of phlorotannin-coated endotracheal tube to prevent tracheal stenosis caused by endotracheal intubation indicates that phlorotannin may be considered as a candidate biomaterial for coating the cuff of endotracheal tubes to prevent tracheal stenosis.