Rapid visual CRISPR assay: a naked-eye colorimetric detection method for nucleic acids based on CRISPR/Cas12a and convolutional neural network (preprint)

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with CRISPR/Cas12a based visual colorimetric assays. Among them, 9 ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for naked-eye read-out of assay results (no transducing component required for visualization). Subsequently, we developed a convolutional neural network algorithm standardize and to automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube towards an instrument-less colorimetric POCT format that requires only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the single-copy detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this novel RAVI-CRISPR system for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen-side testing.

Targeting the Coronavirus Nucleocapsid Protein through GSK-3 Inhibition (preprint)

The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome (MERS-CoV), and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, despite limited overall sequence conservation, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.34 - 0.76], p = 0.001). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type dependent manner. Targeting GSK-3 may therefore provide a new approach to treat COVID-19 and future coronavirus outbreaks.

Clinical characteristics and risk factors for mortality in patients with coronavirus disease 2019 in intensive care unit: a single-center, retrospective, observational study in China (preprint)

Background: Coronavirus disease 2019 (COVID-19) is a potentially life-threatening contagious disease which has spread all over the world. Risk factors for the clinical outcomes of COVID-19 pneumonia in intensive care unit (ICU) have not yet been well determined. Methods: In this retrospective, single-centered, observational study, we consecutively included 47 patients with confirmed COVID-19 who were admitted to the ICU of Leishenshan Hospital in Wuhan, China, from February 24 to April 5, 2020. Clinical characteristics and outcomes were collected and compared between survivors and non-survivors. Multivariable logistic regression was used to explore the risk factors associated with death in patients of COVID-19.Results: The study cohort included 47 adult patients with a median age of 70.55±12.52 years, and 30 (63.8%) patients were men. Totally 15 (31.9%) patients died. Compared with survivors, non-survivors were more likely to develop septic shock (6 [40%] patients vs 3 [9.4%] patients ), disseminated intravascular coagulation (3 [21.4%] vs 0), and had higher score of APACHE II (25.07±8.03 vs 15.56±5.95), CURB-65 (3[2-4] vs 2[1-3]), Sequential Organ Failure Assessment (SOFA) (7[5-9] vs 3[1-6]), higher level of D-dimer (5.74 [2.32-18] vs 2.05 [1.09-4.00] ) and neutrophil count (9.4[7.68-14.54] vs 5.32[3.85-9.34] ). SOFA score (OR 1.47, 1.01–2.13; p=0.0042) and lymphocyte count (OR 0.02, 0.00–0.86; p=0.042) on admission were independently risk factors for mortality. Patients with higher lymphocyte count (>0.63×109/L) and lower SOFA score ≤4 on admission had a significantly well prognosis than those with lower lymphocyte count (≤0.63×109/L) and higher SOFA score >4 in overall survival.Conclusions: Higher SOFA score and lower lymphocyte count on admission were associated with poor prognosis of patients with COVID-19 in ICU. Lymphocyte count may serve as a promising prognostic biomarker.