Application of ultrasensitive assay for SARS-CoV-2 antigen in nasopharynx in the management of COVID-19 patients with comorbidities during the peak of 2022 Shanghai epidemics in a tertiary hospital.

OBJECTIVES: Various comorbidities associated with COVID-19 add up in severity of the disease and obviously prolonged the time for viral clearance. This study investigated a novel ultrasensitive MAGLUMI® SARS-CoV-2 Ag chemiluminescent immunoassay assay (MAG-CLIA) for diagnosis and monitoring the infectivity of COVID-19 patients with comorbid conditions during the pandemic of 2022 Shanghai. METHODS: Analytical performances of the MAG-CLIA were evaluated, including precision, limit of quantitation, linearity and specificity. Nasopharyngeal specimens from 232 hospitalized patients who were SARS-CoV-2 RT-qPCR positive and from 477 healthy donors were included. The longitudinal studies were performed by monitoring antigen concentrations alongside with RT-qPCR results in 14 COVID-19 comorbid participants for up to 22 days. The critical antigen concentration in determining virus infectivity was evaluated at the reference cycle threshold (Ct) of 35. RESULTS: COVID-19 patients were well-identified using an optimal threshold of 0.64 ng/L antigen concentration, with sensitivity and specificity of 95.7% (95% CI: 92.2-97.9%) and 98.3% (95% CI: 96.7-99.3%), respectively, while the Wondfo LFT exhibited those of 34.9% (95% CI: 28.8-41.4%) and 100% (95% CI: 99.23-100%), respectively. The sensitivity of MAG-CLIA remained 91.46% (95% CI: 83.14-95.8%) for the samples with Ct values between 35 and 40. Close dynamic consistence was observed between MAG-CLIA and viral load time series in the longitudinal studies. The critical value of 8.82 ng/L antigen showed adequate sensitivity and specificity in evaluating the infectivity of hospitalized convalescent patients with comorbidities. CONCLUSIONS: The MAG-CLIA SARS-CoV-2 Ag detection is an effective and alternative approach for rapid diagnosis and enables us to evaluate the infectivity of hospitalized convalescent patients with comorbidities.

Gadd45a promotes DNA demethylation through TDG.

Growth arrest and DNA-damage-inducible protein 45 (Gadd45) family members have been implicated in DNA demethylation in vertebrates. However, it remained unclear how they contribute to the demethylation process. Here, we demonstrate that Gadd45a promotes active DNA demethylation through thymine DNA glycosylase (TDG) which has recently been shown to excise 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) generated in Ten-eleven-translocation (Tet)-initiated oxidative demethylation. The connection of Gadd45a with oxidative demethylation is evidenced by the enhanced activation of a methylated reporter gene in HEK293T cells expressing Gadd45a in combination with catalytically active TDG and Tet. Gadd45a interacts with TDG physically and increases the removal of 5fC and 5caC from genomic and transfected plasmid DNA by TDG. Knockout of both Gadd45a and Gadd45b from mouse ES cells leads to hypermethylation of specific genomic loci most of which are also targets of TDG and show 5fC enrichment in TDG-deficient cells. These observations indicate that the demethylation effect of Gadd45a is mediated by TDG activity. This finding thus unites Gadd45a with the recently defined Tet-initiated demethylation pathway.

Plasma SARS-CoV-2 N antigen is a powerful molecular marker for early detection of severe COVID-19 in patients and monitoring disease progression.

BACKGROUND AND AIMS: Clear and effective indicators for early detection of severe coronavirus disease 2019 (COVID-19) are insufficient. We investigated the clinical value of the plasma SARS-CoV-2 N antigen (plasma N antigen) for severe COVID-19 early identification and disease progression monitoring. MATERIALS AND METHODS: A cross-sectional study compared the diagnostic value of plasma N antigen levels detected within two days after hospital admission in 957 patients with COVID-19 during the BA2.2 outbreak in Shanghai (April 6-June 15, 2022). A follow-up study analyzed the plasma N antigen prognostic value in 274 non-severe patients, and a longitudinal study evaluated its continuous monitoring value in 16 patients with COVID-19 grade changes. RESULTS: Plasma N antigen concentrations were significantly higher in severely ill than in non-severely ill patients. The plasma N antigen was superior to nasopharyngeal nucleic acid CT values and established COVID-19 blood biomarkers in identifying severe COVID-19. Patients with high plasma N-antigen concentrations at initial admission were more prone to developing severe COVID-19. The changes in plasma N antigen concentrations were consistent with disease progression. Two logistic regression models, including and excluding plasma N antigen, were established, with model 1 (including plasma N antigen) (AUC = 0.971, 0.958-0.980) yielding a better diagnostic value for severe COVID-19 than Model 2 (plasma N antigen excluded). CONCLUSION: The plasma N antigen is superior to nasopharyngeal nucleic acids and established COVID-19 blood biomarkers for severe COVID-19 early recognition and progression monitoring, enabling the most accurate patient triaging and efficient utilization of medical resources.

Promoter G-quadruplex folding precedes transcription and is controlled by chromatin.

BACKGROUND: Four-stranded G-quadruplexes (G4s) are DNA secondary structures in the human genome that are primarily found in active promoters associated with elevated transcription. Here, we explore the relationship between the folding of promoter G4s, transcription and chromatin state. RESULTS: Transcriptional inhibition by DRB or by triptolide reveals that promoter G4 formation, as assessed by G4 ChIP-seq, does not depend on transcriptional activity. We then show that chromatin compaction can lead to loss of promoter G4s and is accompanied by a corresponding loss of RNA polymerase II (Pol II), thus establishing a link between G4 formation and chromatin accessibility. Furthermore, pre-treatment of cells with a G4-stabilising ligand mitigates the loss of Pol II at promoters induced by chromatin compaction. CONCLUSIONS: Overall, our findings show that G4 folding is coupled to the establishment of accessible chromatin and does not require active transcription.

Single-molecule visualization of DNA G-quadruplex formation in live cells.

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) is coupled to altered gene expression. However, approaches that allow us to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells reveal that G4s fluctuate between folded and unfolded states. We also demonstrate that G4 formation in live cells is cell-cycle-dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4 formation in living cells.

The RNA-binding protein LARP4 regulates cancer cell migration and invasion.

LARP4 is a La-related RNA-binding protein implicated in regulating mRNA translation, which interacts with poly(A)-binding protein (PABP). We previously identified LARP4 in an RNAi screen as one of several genes that regulate the shape of PC3 prostate cancer cells. Here we show that LARP4 depletion induces cell elongation in PC3 cells and MDA-MB-231 breast cancer cells. LARP4 depletion increases cell migration and invasion, as well as inducing invasive cell protrusions in 3D Matrigel. Conversely, LARP4 over-expression reduces cell elongation and increases cell circularity. LARP4 mutations are found in a variety of cancers. Introduction of some of these cancer-associated mutations, including a truncation mutant, into LARP4 enhances its effects on cell morphology. The truncation mutant shows enhanced interaction with PABP. We propose that LARP4 inhibits migration and invasion of cancer cells, and that some cancer-associated mutations stimulate these effects of LARP4. © 2016 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.