Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a.

CRISPR-based assays for the detection of nucleic acids are highly specific, yet they are not fast, sensitive or easy to use. Here we report a one-step fluorescence assay for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in nasopharyngeal samples, with a sample-to-answer time of less than 20 minutes and a sensitivity comparable to that of quantitative real-time PCR with reverse transcription (RT-qPCR). The assay uses suboptimal protospacer adjacent motifs, allowing for flexibility in the design of CRISPR RNAs and slowing down the kinetics of Cas12a-mediated collateral cleavage of fluorescent DNA reporters and cis cleavage of substrates, which leads to stronger fluorescence owing to the accumulation of amplicons generated by isothermal recombinase polymerase amplification. In a set of 204 nasopharyngeal samples with RT-qPCR cycle thresholds ranging from 18.1 to 35.8, the assay detected SARS-CoV-2 with a sensitivity of 94.2% and a specificity of 100%, without the need for RNA extraction. Rapid and sensitive assays for nucleic acid testing in one pot that allow for flexibility in assay design may aid the development of reliable point-of-care nucleic acid testing.

SARS-CoV-2 Membrane Glycoprotein M Triggers Apoptosis With the Assistance of Nucleocapsid Protein N in Cells.

The pandemic of COVID-19 by SARS-CoV-2 has become a global disaster. However, we still don't know how specific SARS-CoV-2-encoded proteins contribute to viral pathogenicity. We found that SARS-CoV-2-encoded membrane glycoprotein M could induce caspase-dependent apoptosis via interacting with PDK1 and inhibiting the activation of PDK1-PKB/Akt signaling. Our investigation further revealed that SARS-CoV-2-encoded nucleocapsid protein N could specifically enhance the M-induced apoptosis via interacting with both M and PDK1, therefore strengthening M-mediated attenuation of PDK1-PKB/Akt interaction. Furthermore, when the M-N interaction was disrupted via certain rationally designed peptides, the PDK1-PKB/Akt signaling was restored, and the boosting activity of N on the M-triggered apoptosis was abolished. Overall, our findings uncovered a novel mechanism by which SARS-CoV-2-encoded M triggers apoptosis with the assistance of N, which expands our understanding of the two key proteins of SARS-CoV-2 and sheds light on the pathogenicity of this life-threatening virus.

Multi-omic profiling of plasma reveals molecular alterations in children with COVID-19.

Rationale: Children usually develop less severe symptoms responding to Coronavirus Disease 2019 (COVID-19) than adults. However, little is known about the molecular alterations and pathogenesis of COVID-19 in children. Methods: We conducted plasma proteomic and metabolomic profilings of the blood samples of a cohort containing 18 COVID-19-children with mild symptoms and 12 healthy children, which were enrolled from hospital admissions and outpatients, respectively. Statistical analyses were performed to identify molecules specifically altered in COVID-19-children. We also developed a machine learning-based pipeline named inference of biomolecular combinations with minimal bias (iBM) to prioritize proteins and metabolites strongly altered in COVID-19-children, and experimentally validated the predictions. Results: By comparing to the multi-omic data in adults, we identified 44 proteins and 249 metabolites differentially altered in COVID-19-children against healthy children or COVID-19-adults. Further analyses demonstrated that both deteriorative immune response/inflammation processes and protective antioxidant or anti-inflammatory processes were markedly induced in COVID-19-children. Using iBM, we prioritized two combinations that contained 5 proteins and 5 metabolites, respectively, each exhibiting a total area under curve (AUC) value of 100% to accurately distinguish COVID-19-children from healthy children or COVID-19-adults. Further experiments validated that all the 5 proteins were up-regulated upon coronavirus infection. Interestingly, we found that the prioritized metabolites inhibited the expression of pro-inflammatory factors, and two of them, methylmalonic acid (MMA) and mannitol, also suppressed coronaviral replication, implying a protective role of these metabolites in COVID-19-children. Conclusion: The finding of a strong antagonism of deteriorative and protective effects provided new insights on the mechanism and pathogenesis of COVID-19 in children that mostly underwent mild symptoms. The identified metabolites strongly altered in COVID-19-children could serve as potential therapeutic agents of COVID-19.

Plasma metabolomic and lipidomic alterations associated with COVID-19.

The pandemic of the coronavirus disease 2019 (COVID-19) has become a global public health crisis. The symptoms of COVID-19 range from mild to severe, but the physiological changes associated with COVID-19 are barely understood. In this study, we performed targeted metabolomic and lipidomic analyses of plasma from a cohort of patients with COVID-19 who had experienced different symptoms. We found that metabolite and lipid alterations exhibit apparent correlation with the course of disease in these patients, indicating that the development of COVID-19 affected their whole-body metabolism. In particular, malic acid of the TCA cycle and carbamoyl phosphate of the urea cycle result in altered energy metabolism and hepatic dysfunction, respectively. It should be noted that carbamoyl phosphate is profoundly down-regulated in patients who died compared with patients with mild symptoms. And, more importantly, guanosine monophosphate (GMP), which is mediated not only by GMP synthase but also by CD39 and CD73, is significantly changed between healthy subjects and patients with COVID-19, as well as between the mild and fatal cases. In addition, dyslipidemia was observed in patients with COVID-19. Overall, the disturbed metabolic patterns have been found to align with the progress and severity of COVID-19. This work provides valuable knowledge about plasma biomarkers associated with COVID-19 and potential therapeutic targets, as well as an important resource for further studies of the pathogenesis of COVID-19.

Imaging Mass Cytometric Analysis of Postmortem Tissues Reveals Dysregulated Immune Cell and Cytokine Responses in Multiple Organs of COVID-19 Patients.

SARS-coronavirus-2-induced immune dysregulation and inflammatory responses are involved in the pathogenesis of coronavirus disease-2019 (COVID-19). However, very little is known about immune cell and cytokine alterations in specific organs of COVID-19 patients. Here, we evaluated immune cells and cytokines in postmortem tissues, i.e., lungs, intestine, liver, kidneys, and spleen of three patients with COVID-19. Imaging mass cytometry revealed monocyte, macrophage, and dendritic cell (DC) infiltration in the lung, intestine, kidney, and liver tissues. Moreover, in patients with COVID-19, natural killer T cells infiltrated the liver, lungs, and intestine, whereas B cells infiltrated the kidneys, lungs, and intestine. CD11b+ macrophages and CD11c+ DCs also infiltrated the lungs and intestine, a phenomenon that was accompanied by overproduction of the immunosuppressive cytokine interleukin (IL)-10. However, CD11b+ macrophages and CD11c+ DCs in the lungs or intestine of COVID-19 patients did not express human leukocyte antigen DR isotype. In contrast, tumor necrosis factor (TNF)-α expression was higher in the lungs, intestine, liver, and kidneys, but not in the spleen, of all COVID-19 patients (compared to levels in controls). Collectively, these findings suggested that IL-10 and TNF-α as immunosuppressive and pro-inflammatory agents, respectively,-might be prognostic and could serve as therapeutic targets for COVID-19.

SARS-Coronavirus-2 Nsp13 Possesses NTPase and RNA Helicase Activities That Can Be Inhibited by Bismuth Salts.

The ongoing outbreak of Coronavirus Disease 2019 (COVID-19) has become a global public health emergency. SARS-coronavirus-2 (SARS-CoV-2), the causative pathogen of COVID-19, is a positive-sense single-stranded RNA virus belonging to the family Coronaviridae. For RNA viruses, virus-encoded RNA helicases have long been recognized to play pivotal roles during viral life cycles by facilitating the correct folding and replication of viral RNAs. Here, our studies show that SARS-CoV-2-encoded nonstructural protein 13 (nsp13) possesses the nucleoside triphosphate hydrolase (NTPase) and RNA helicase activities that can hydrolyze all types of NTPs and unwind RNA helices dependently of the presence of NTP, and further characterize the biochemical characteristics of these two enzymatic activities associated with SARS-CoV-2 nsp13. Moreover, we found that some bismuth salts could effectively inhibit both the NTPase and RNA helicase activities of SARS-CoV-2 nsp13 in a dose-dependent manner. Thus, our findings demonstrate the NTPase and helicase activities of SARS-CoV-2 nsp13, which may play an important role in SARS-CoV-2 replication and serve as a target for antivirals.

Plasma Proteomics Identify Biomarkers and Pathogenesis of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic is a global public health crisis. However, little is known about the pathogenesis and biomarkers of COVID-19. Here, we profiled host responses to COVID-19 by performing plasma proteomics of a cohort of COVID-19 patients, including non-survivors and survivors recovered from mild or severe symptoms, and uncovered numerous COVID-19-associated alterations of plasma proteins. We developed a machine-learning-based pipeline to identify 11 proteins as biomarkers and a set of biomarker combinations, which were validated by an independent cohort and accurately distinguished and predicted COVID-19 outcomes. Some of the biomarkers were further validated by enzyme-linked immunosorbent assay (ELISA) using a larger cohort. These markedly altered proteins, including the biomarkers, mediate pathophysiological pathways, such as immune or inflammatory responses, platelet degranulation and coagulation, and metabolism, that likely contribute to the pathogenesis. Our findings provide valuable knowledge about COVID-19 biomarkers and shed light on the pathogenesis and potential therapeutic targets of COVID-19.