Production of a versatile SARS-CoV-2 main protease biosensor based on a dimerization-dependent red fluorescent protein.

we designed a functionally active Mpro biosensor based on a dimerization-dependent red fluorescent protein (ddRFP) for the evaluation of Mpro inhibitors in vitro. This study provides an affordable strategy for rapid production of a versatile ddRFP biosensor, which would be a useful tool for the measurement and quantification of Mpro inhibitors This article is protected by copyright. All rights reserved.

Protocol for high-throughput screening of SARS-CoV-2 main protease inhibitors using a robust fluorescence polarization assay.

Discovery of efficacious antiviral agents targeting SARS-CoV-2 main protease (Mpro) is of the highest importance to fight against COVID-19. Here, we describe a simple protocol for high-throughput screening of Mpro inhibitors using a robust fluorescence polarization (FP) assay. Candidate Mpro inhibitors from large compound libraries could be rapidly identified by monitoring the change of millipolarization unit value. This affordable FP assay can be modified to screen antiviral agents targeting virus protease. For complete details on the use and execution of this protocol, please refer to Li et al. (2022), Yan et al. (2021), and Yan et al. (2022c).

Protocol for high-throughput screening of SARS-CoV-2 main protease inhibitors using a robust fluorescence polarization assay

Discovery of efficacious antiviral agents targeting SARS-CoV-2 main protease (Mpro) is of the highest importance to fight against COVID-19. Here, we describe a simple protocol for high-throughput screening of Mpro inhibitors using a robust fluorescence polarization (FP) assay. Candidate Mpro inhibitors from large compound libraries could be rapidly identified by monitoring the change of millipolarization unit value. This affordable FP assay can be modified to screen antiviral agents targeting viral protease. Graphical Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Development and validation of chest CT-based imaging biomarkers for early stage COVID-19 screening

Coronavirus Disease 2019 (COVID-19) is currently a global pandemic, and early screening is one of the key factors for COVID-19 control and treatment. Here, we developed and validated chest CT-based imaging biomarkers for COVID-19 patient screening from two independent hospitals with 419 patients. We identified the vasculature-like signals from CT images and found that, compared to healthy and community acquired pneumonia (CAP) patients, COVID-19 patients display a significantly higher abundance of these signals. Furthermore, unsupervised feature learning led to the discovery of clinical-relevant imaging biomarkers from the vasculature-like signals for accurate and sensitive COVID-19 screening that have been double-blindly validated in an independent hospital (sensitivity: 0.941, specificity: 0.920, AUC: 0.971, accuracy 0.931, F1 score: 0.929). Our findings could open a new avenue to assist screening of COVID-19 patients.

A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors.

The global scourge of COVID-19 is a serious threat to public health, but effective therapies remain very limited for this disease. Therefore, the discovery of novel antiviral agents is urgently needed to fight against COVID-19. In the lifecycle of SARS-CoV-2, the causing pathogen of COVID-19, papain-like protease (PLpro) is responsible for the cleavage of polyprotein into functional units as well as immune evasion of vaccines. Hence, PLpro has been regarded as an attractive target to develop antiviral agents. Herein, we first developed a robust and simple sandwich-like fluorescence polarization (FP) screening assay for the discovery of PLpro inhibitors, and identified anacardic acid as a novel competitive inhibitor against PLpro in vitro with an IC50 value of 24.26 ± 0.4 µM. This reliable FP screening assay could provide a prospective avenue for rapid discovery of antiviral agents targeting PLpro in a large-scale screening.

Ginkgolic acid and anacardic acid are reversible inhibitors of SARS-CoV-2 3-chymotrypsin-like protease.

Because of the emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in different regions of the world, the battle with infectious coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 has been seesawing. Therefore, the identification of antiviral drugs is of particular importance. In order to rapidly identify inhibitors for SARS-CoV-2 3-chymotrypsin-like protease (3CLpro), an enzyme essential for viral replication, we combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) and developed a novel sandwich-like FP screening assay. Through high-throughput screening, two hits of 3CLpro inhibitors, ginkgolic acid (GA) and anacardic acid (AA) were identified, which showed IC50 values of 11.29 ± 0.48 and 12.19 ± 0.50 µM, respectively. Their binding modes were evaluated by HPLC-Q-TOF-MS. There was no mass increase detected for SARS-CoV-2 3CLpro incubated with either GA or AA, indicating the absence of covalent adducts. The kinetic analysis clearly demonstrated that both GA and AA inhibit SARS-CoV-2 3CLpro via reversible and mixed-inhibition manner. Our results argue against conclusion that GA and AA act as irreversible and covalent inhibitors against SARS-CoV-2 3CLpro, which is based on the studies by Chen et al.

[Identifying SARS-CoV-2 main protease inhibitors by a novel sandwich-like fluorescence polarization screening assay].

SARS-CoV-2 main protease (Mpro) is responsible for polyprotein cleavage to release non-structural proteins (nsps) for viral genomic RNA replication, and its homologues are absent in human cells. Therefore, Mpro has been regarded as one of the ideal drug targets for the treatment of coronavirus disease 2019 (COVID-19). In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel sandwich-like FP screening assay for quick discovery of SARS-CoV-2 Mpro inhibitors from a natural product library. With this screening assay, anacardic acid (AA) and 1, 2, 3, 4, 6-O-pentagalloylglucose (PGG) were found to be the competitive inhibitor and mixed-type inhibitor targeting Mpro, respectively. Importantly, our results showed that the majority of the reported Mpro inhibitors are promiscuous cysteine inhibitors that are not specific to Mpro. In summary, this novel sandwich-like FP screening assay is simple, sensitive, and robust, which is ideal for large-scale screening. Natural products AA and PGG will be the promising lead compounds for generating more potent antiviral agents targeting Mpro, and the stringent hit validation at the early stage of drug discovery is urgently needed.

[Discovery of SARS-CoV-2 main protease inhibitors using an optimized FRET-based high-throughput screening assay].

For rapid discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors, an optimized fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay was developed. The recombinant Mpro was expressed in Escherichia coli Rosetta (DE3) cells and the specific activity of purified Mpro was assessed by a FERT assay using a fluorescently labeled substrate. Subsequently, the reaction buffer, working concentration of Mpro, incubation temperature and length, and DMSO tolerance were systematically optimized. The Mpro was solubly expressed in E. coli cells and exhibited an expected enzymatic activity (40 000 U/mg) in a FRET assay. Through these systematic optimizations, we selected 0.4 µmol/L Mpro and 5 µmol/L FRET substrate as the optimal working concentrations in this FRET screening assay, and a high Z' factor of 0.79 was achieved. More importantly, the addition of reducing reagent 1, 4-dithiothreitol in reaction buffer is necessary to faithfully assess the reliability of the screening assay. Using this assay, plumbagin (PLB) and ginkgolic acid (GA) were identified as potential Mpro inhibitors in vitro from a natural product library. In summary, we developed an optimized FRET-based HTS assay for the discovery of Mpro inhibitors, and PLB and GA could serve as the promissing lead compounds to generate more potent antiviral agents targeting SARS-CoV-2 Mpro.

Mapping Building-Based Spatiotemporal Distributions of Carbon Dioxide Emission: A Case Study in England.

The spatiotemporal inventory of carbon dioxide (CO2) emissions from the building sector is significant for formulating regional and global warming mitigation policies. Previous studies have attempted to use energy consumption models associated with field investigations to estimate CO2 emissions from buildings at local scales, or they used spatial proxies to downscale emission sources from large geographic units to grid cells for larger scales. However, mapping the spatiotemporal distributions of CO2 emissions on a large scale based on buildings remains challenging. Hence, we conducted a case study in England in 2015, wherein we developed linear regression models to analyze monthly CO2 emissions at the building scale by integrating the Emissions Database for Global Atmospheric Research, building data, and Visible Infrared Imaging Radiometer Suite night-time lights images. The results showed that the proposed model that considered building data and night-time light imagery achieved the best fit. Fine-scale spatial heterogeneity was observed in the distributions of building-based CO2 emissions compared to grid-based emission maps. In addition, we observed seasonal differences in CO2 emissions. Specifically, buildings emitted significantly more CO2 in winter than in summer in England. We believe our results have great potential for use in carbon neutrality policy making and climate monitoring.

Comparison of Laboratory Data between Children with Kawasaki Disease and COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has been an emerging, rapidly evolving situation in China since late 2019 and has even become a worldwide pandemic. The first case of severe childhood novel coronavirus pneumonia in China was reported in March 2020 in Wuhan. The severity differs between adults and children, with lower death rates and decreased severity for individuals under the age of 20 years. Increased cases of Kawasaki disease (KD) have been reported from New York City and some areas of Italy and the U.K., with almost a 6-10 times increase when compared to previous years. We conducted this study to compare characteristics and laboratory data between KD and COVID-19 in children. METHODS: We obtained a total of 24 children with COVID-19 from a literature review and 268 KD cases from our hospital via retrospective chart review. RESULTS: We found that patients with KD have higher levels of white blood cells (WBCs), platelets, neutrophil percentage, C-reactive protein (CRP), procalcitonin, and aspartate aminotransferase (AST) and a higher body temperature, while patients with COVID-19 have a higher age, hemoglobin levels, and lymphocyte percentage. After performing multiple logistic regression analysis, we found that age, WBCs, platelets, procalcitonin, and AST are identical markers for distinguishing COVID-19 from KD in children. CONCLUSION: In this COVID-19 pandemic period, clinicians should pay attention to children with COVID-19 infection when high WBC, platelet, procalcitonin, and AST values are present in order to provide early diagnosis for KD or multisystem inflammatory syndrome in children (MIS-C).

Alzheimer's-like signaling in brains of COVID-19 patients.

INTRODUCTION: The mechanisms that lead to cognitive impairment associated with COVID-19 are not well understood. METHODS: Brain lysates from control and COVID-19 patients were analyzed for oxidative stress and inflammatory signaling pathway markers, and measurements of Alzheimer's disease (AD)-linked signaling biochemistry. Post-translational modifications of the ryanodine receptor/calcium (Ca2+ ) release channels (RyR) on the endoplasmic reticuli (ER), known to be linked to AD, were also measured by co-immunoprecipitation/immunoblotting of the brain lysates. RESULTS: We provide evidence linking SARS-CoV-2 infection to activation of TGF-ß signaling and oxidative overload. The neuropathological pathways causing tau hyperphosphorylation typically associated with AD were also shown to be activated in COVID-19 patients. RyR2 in COVID-19 brains demonstrated a "leaky" phenotype, which can promote cognitive and behavioral defects. DISCUSSION: COVID-19 neuropathology includes AD-like features and leaky RyR2 channels could be a therapeutic target for amelioration of some cognitive defects associated with SARS-CoV-2 infection and long COVID.

Development of a simple and miniaturized sandwich-like fluorescence polarization assay for rapid screening of SARS-CoV-2 main protease inhibitors.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present. RESULTS: In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC50 value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model. CONCLUSIONS: This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.

Human Coronavirus Infections and Pregnancy.

Human coronavirus (HCoV) causes potentially fatal respiratory disease. Pregnancy is a physiological state that predisposes women to viral infection. In this review, we aim to present advances in the pathogenesis, clinical features, diagnosis, and treatment in HCoV in pregnancy. We retrieved information from the Pubmed database up to June 2020, using various search terms and relevant words, including coronaviruses, severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, 2019 coronavirus disease, and pregnancy. Both basic and clinical studies were selected. We found no evidence that pregnant women are more susceptible to HCoV infection or that those with HCoV infection are more prone to developing severe pneumonia. There is also no confirmed evidence of vertical mother-to-child transmission of HcoV infection during maternal HCoV infection. Those diagnosed with infection should be promptly admitted to a negative-pressure isolation ward, preferably in a designated hospital with adequate facilities and multi-disciplinary expertise to manage critically ill obstetric patients. Antiviral treatment has been routinely used to treat pregnant women with HCoV infection. The timing and mode of delivery should be individualized, depending mainly on the clinical status of the patient, gestational age, and fetal condition. Early cord clamping and temporary separation of the newborn for at least 2 weeks is recommended. All medical staff caring for patients with HCoV infection should use personal protective equipment. This review highlights the advances in pathogenesis, maternal-fetal outcome, maternal-fetal transmission, diagnosis and treatment in HCoV including severe acute respiratory syndrome coronavirus, Middle East respiratory syndrome coronavirus, and coronavirus disease 2019 in pregnancy.

Correlation between ground-glass opacity on pulmonary CT and the levels of inflammatory cytokines in patients with moderate-to-severe COVID-19 pneumonia.

Objectives: Comparative analysis of laboratory data in moderate-to-severe COVID-19 patients presenting with or without ground-glass opacities (GGOs). Methods: This retrospective study examined 61 patients with moderate-to-severe COVID-19, as defined by the report of the WHO-China Joint Mission on COVID-19. All patients were admitted to the Department of Infectious Diseases, Wuhan Union Hospital from Dec 28, 2019 to Feb 22, 2020 and classified into a GGO group or a non-GGO group based on CT results. The clinical characteristics and laboratory data of the two groups were compared. Data were analyzed using univariate and multivariate analysis, and using receiver operating characteristic (ROC) analysis. Results: Forty-five patients were in the GGO group (73.8%, 21 females, 24 males, mean age 54.8±17.8 years) and 16 were in the non-GGO group (26.2%, 11 females, 5 males, mean age 53±14.9 years). The levels of IL-2, IL-4, and IFN-γ were greater in the GGO group (all P<0.05). ROC analysis indicated that an elevated level of IL-2 was a good predictor of GGO (area under the curve: 0.716, optimal cutoff: 3.205 pg/mL, 53.8% sensitivity, 87.5% specificity, p<0.05). Multivariate analysis showed that IL-2 level was a significant and independent risk factor for lung GGO (OR: 8.167; 95% CI: 1.63, 40.8; P<0.05). Conclusions: There were correlations between GGO in the lungs of patients with moderate-to-severe COVID-19 and the levels of IL-2, IL-4, and INF-γ. IL-2 was a significant and independent risk factor for GGO. These findings provide a basis for studying the mechanism of pulmonary lesions in COVID-19 patients.

[Optimization of expression conditions and determination the proteolytic activity of codon-optimized SARS-CoV-2 main protease in Escherichia coli].

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.

A simple, SIR-like but individual-based epidemic model: Application in comparison of COVID-19 in New York City and Wuhan.

In this study, an individual-based epidemic model, considering latent-infectious-recovery periods, is presented. The analytic solution of the model in the form of recursive formulae with a time-dependent transmission coefficient is derived and implanted in Excel. The simulated epidemic curves from the model fit very well with the daily reported cases of COVID-19 in Wuhan, China and New York City (NYC), USA. These simulations show that the transmission rate of NYC's COVID-19 is nearly 30% greater than the transmission rate of Wuhan's COVID-19, and that the actual number of cumulative infected people in NYC is around 9 times the reported number of cumulative COVID-19 cases in NYC. Results from this study also provide important information about latent period, infectious period and lockdown efficiency.

Risk factors for delayed negative conversion of SARS-CoV-2 in patients with COVID-19 pneumonia: a retrospective cohort study.

The epidemic of coronavirus disease 2019 (COVID-19) began in China and had spread rapidly to many other countries. This study aimed to identify risk factors associated with delayed negative conversion of SARS-CoV-2 in COVID-19 patients. In this retrospective single-centre study, we included 169 consecutive patients with confirmed COVID-19 in Zhongnan Hospital of Wuhan University from 15th January to 2nd March. The cases were divided into two groups according to the median time of SARS-CoV-2 negative conversion. The differences between groups were compared. In total, 169 patients had a median virus negative conversion time of 18 days (interquartile range: 11-25) from symptom onset. Compared with the patients with short-term negative conversion, those with long-term conversion had an older age, higher incidence of comorbidities, chief complaints of cough and chest distress/breath shortness and severer illness on admission, higher level of leucocytes, neutrophils, aspartate aminotransferase, creatine kinase and erythrocyte sedimentation rate (ESR), lower level of CD3+CD4+ lymphocytes and albumin and more likely to receive mechanical ventilation. In multivariate analysis, cough, leucocytes, neutrophils and ESR were positively correlated with delayed virus negative conversion, and CD3+CD4+ lymphocytes were negatively correlated. The integrated indicator of leucocytes, neutrophils and CD3+CD4+ lymphocytes showed a good performance in predicting the negative conversion within 2 weeks (area under ROC curve (AUC) = 0.815), 3 weeks (AUC = 0.804), 4 weeks (AUC = 0.812) and 5 weeks (AUC = 0.786). In conclusion, longer quarantine periods might be more justified for COVID-19 patients with cough, higher levels of leucocytes, neutrophils and ESR and lower levels of CD3+CD4+ lymphocytes.

An online tool for predicting the prognosis of cancer patients with SARS-CoV-2 infection: a multi-center study.

PURPOSE: During the 2019 coronavirus disease (COVID-19) pandemic, oncologists face new challenges, and they need to adjust their cancer management strategies as soon as possible to reduce the risk of SARS-CoV-2 infection and tumor recurrence. However, data on cancer patients with SARS-CoV-2 infection remains scarce. METHODS: We conducted a retrospective study on 223 cancer patients with SARS-CoV-2 from 26 hospitals in Hubei, China. An individualized nomogram was constructed based on multivariate Cox analysis. Considering the convenience of the nomogram application, an online tool was also created. The predictive performance and clinical application of nomogram were verified by C-index, calibration curve and decision curve analysis (DCA). RESULTS: Among cancer patients with SARS-CoV-2, there were significant differences in clinical characteristics between survivors and non-survivors, and compared with patients with solid tumors including lung cancer, patients with hematological malignancies had a worse prognosis. Male, dyspnea, elevated PCT, increased heart rate, elevated D-dimers, and decreased platelets were risk factors for these patients. Furthermore, a good prediction performance of the online tool (dynamic nomogram: https://covid-19-prediction-tool.shinyapps.io/DynNomapp/ ) was also fully demonstrated with the C-indexes of 0.841 (95% CI 0.782-0.900) in the development cohort and 0.780 (95% CI 0.678-0.882) in the validation cohort. CONCLUSION: Overall, cancer patients with SARS-CoV-2 had unique clinical features, and the established online tool could guide clinicians to predict the prognosis of patients during the COVID-19 epidemic and to develop more rational treatment strategies for cancer patients.