In canonical Wnt/β-catenin signaling pathway, β-catenin/TCF4 (T-cell factor 4) interaction plays an important role in the pathogenesis and development of non-small cell lung cancer (NSCLC), and it is tightly associated with the proliferation, chemoresistance, recurrence and metastasis of NSCLC. Therefore, suppressing β-catenin/TCF4 interaction in Wnt/β-catenin signaling pathway would be a new therapeutic avenue against NSCLC metastasis. In this study, considering the principle of enzyme-linked immunosorbent assay (ELISA), an optimized high-throughput screening (HTS) assay was developed for the discovery of β-catenin/TCF4 interaction antagonists. Subsequently, this ELISA-like screening assay was performed using 2 μg/mL GST-TCF4 βBD and 0.5 μg/mL β-catenin, then a high Z' factor of 0.83 was achieved. A pilot screening of a natural product library using this ELISA-like screening assay identified plumbagin as a potential β-catenin/TCF4 interaction antagonist. Plumbagin remarkably inhibited the proliferation of A549, H1299, MCF7 and SW480 cell lines. More importantly, plumbagin significantly suppressed the β-catenin-responsive transcription in TOPFlash assay. In short, this newly developed ELISA-like screening assay will be vital for the rapid screening of novel Wnt inhibitors targeting β-catenin/TCF4 interaction, and this interaction is a potential anticancer target of plumbagin in vitro.
Carcinoma, Non-Small-Cell Lung ; Cell Line, Tumor ; Enzyme-Linked Immunosorbent Assay ; High-Throughput Screening Assays ; Humans ; Lung Neoplasms ; Transcription Factor 4/genetics* ; beta Catenin/genetics*

The main protease (Mpro) of SARS-CoV-2 is a highly conserved and mutation-resistant coronaviral enzyme, which plays a pivotal role in viral replication, making it an ideal target for the development of novel broad-spectrum anti-coronaviral drugs. In this study, a codon-optimized Mpro gene was cloned into pET-21a and pET-28a expression vectors. The recombinant plasmids were transformed into E. coli Rosetta(DE3) competent cells and the expression conditions were optimized. The highly expressed recombinant proteins, Mpro and Mpro-28, were purified by HisTrapTM chelating column and its proteolytic activity was determined by a fluorescence resonance energy transfer (FRET) assay. The FRET assay showed that Mpro exhibits a desirable proteolytic activity (25 000 U/mg), with Km and kcat values of 11.68 μmol/L and 0.037/s, respectively. The specific activity of Mpro is 25 times that of Mpro-28, a fusion protein carrying a polyhistidine tag at the N and C termini, indicating additional residues at the N terminus of Mpro, but not at the C terminus, are detrimental to its proteolytic activity. The preparation of active SARS-CoV-2 Mpro through codon-optimization strategy might facilitate the development of the rapid screening assays for the discovery of broad-spectrum anti-coronaviral drugs targeting Mpro.
COVID-19 ; Codon/genetics* ; Cysteine Endopeptidases/genetics* ; Escherichia coli/genetics* ; Humans ; Peptide Hydrolases ; SARS-CoV-2 ; Viral Nonstructural Proteins/genetics*

Objective To explore the clinical features,diagnostic methods,and treatment strategies for severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)encephalitis confirmed through cerebrospinal fluid(CSF)analysis.Methods The clinical data of patients diagnosed with SARS-CoV-2 encephalitis through CSF analysis in the Neurology Intensive Care Unit of the First Affiliated Hospital of Nanchang University from March 2022 to March 2023 were collected.Additionally,the relevant literature published in both domestic and international databases was analyzed and synthesized.Results The main neurological manifestations of five cases included decreased consciousness(5/5),psychiatric disorder(2/5),seizures(2/5),quadriplegia(1/5),and headaches(1/5).Two cases had abnormal brain magnetic resonance imaging(MRI)changes,involving the temporal lobe,insular lobe,thalamus,hippocampus,and pons.Additionally,CSF analysis showed mildly elevated protein levels in two cases.Next-generation sequencing(NGS)of the CSF identified SARS-CoV-2 in all five cases(sequence:41-1620),and human herpesvirus 1 in one case(sequence:21).The treatment regimen for all cases included antiviral therapy,three were additionally treated with glucocorticoids and one received immunoglobulin therapy.All cases achieved a favorable outcome(mRS:0-2).Conclusion SARS-CoV-2 has the potential to induce encephalitis/meningitis due to its neurotropic nature.The consideration of this condition is warranted in patients with relevant epidemiological history and symptoms related to the central nervous system.CSF NGS serves as a valuable tool for early diagnosis,while active antiviral therapy and immunotherapy may improve patient outcomes.