CRISPR-Cas12a Coupled with DNA Nanosheet-Amplified Fluorescence Anisotropy for Sensitive Detection of Biomolecules.

DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong trans-cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the trans-cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the trans-cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.

Innate Immune Evasion of Porcine Epidemic Diarrhea Virus through Degradation of the FBXW7 Protein via the Ubiquitin-Proteasome Pathway.

Porcine epidemic diarrhea virus (PEDV) causes a porcine disease associated with swine epidemic diarrhea. Different antagonistic strategies have been identified, and the mechanism by which PEDV infection impairs the production of interferon (IFN) and delays the activation of the IFN response to escape host innate immunity has been determined, but the pathogenic mechanisms of PEDV infection remain enigmatic. Our preliminary results revealed that endogenous F-box and WD repeat domain-containing 7 (FBXW7) protein, the substrate recognition component of the SCF-type E3 ubiquitin ligase, is downregulated in PEDV-infected Vero E6 cells, according to the results from an isobaric tags for relative and absolute quantification (iTRAQ) analysis. Overexpression of FBXW7 in target cells makes them more resistant to PEDV infection, whereas ablation of FBXW7 expression by small interfering RNA (siRNA) significantly promotes PEDV infection. In addition, FBXW7 was verified as an innate antiviral factor capable of enhancing the expression of RIG-I and TBK1, and it was found to induce interferon-stimulated genes (ISGs), which led to an elevated antiviral state of the host cells. Moreover, we revealed that PEDV nonstructural protein 2 (nsp2) interacts with FBXW7 and targets FBXW7 for degradation through the K48-linked ubiquitin-proteasome pathway. Consistent with the results proven in vitro, FBXW7 reduction was also confirmed in different intestinal tissues from PEDV-infected specific-pathogen-free (SPF) pigs. Taken together, the data indicated that PEDV has evolved with a distinct antagonistic strategy to circumvent the host antiviral response by targeting the ubiquitin-proteasome-mediated degradation of FBXW7. Our findings provide novel insights into PEDV infection and pathogenesis. IMPORTANCE To counteract the host antiviral defenses, most viruses, including coronaviruses, have evolved with diverse strategies to dampen host IFN-mediated antiviral response, by interfering with or evading specific host regulators at multiple steps of this response. In this study, a novel antagonistic strategy was revealed showing that PEDV infection could circumvent the host innate response by targeted degradation of endogenous FBXW7 in target cells, a process that was verified to be a positive modulator for the host innate immune system. Degradation of FBXW7 hampers host innate antiviral activation and facilitates PEDV replication. Our findings reveal a new mechanism exploited by PEDV to suppress the host antiviral response.

Artificial intelligence model on chest imaging to diagnose COVID-19 and other pneumonias: A systematic review and meta-analysis.

Objectives: When diagnosing Coronavirus disease 2019(COVID-19), radiologists cannot make an accurate judgments because the image characteristics of COVID-19 and other pneumonia are similar. As machine learning advances, artificial intelligence(AI) models show promise in diagnosing COVID-19 and other pneumonias. We performed a systematic review and meta-analysis to assess the diagnostic accuracy and methodological quality of the models. Methods: We searched PubMed, Cochrane Library, Web of Science, and Embase, preprints from medRxiv and bioRxiv to locate studies published before December 2021, with no language restrictions. And a quality assessment (QUADAS-2), Radiomics Quality Score (RQS) tools and CLAIM checklist were used to assess the quality of each study. We used random-effects models to calculate pooled sensitivity and specificity, I2 values to assess heterogeneity, and Deeks' test to assess publication bias. Results: We screened 32 studies from the 2001 retrieved articles for inclusion in the meta-analysis. We included 6737 participants in the test or validation group. The meta-analysis revealed that AI models based on chest imaging distinguishes COVID-19 from other pneumonias: pooled area under the curve (AUC) 0.96 (95 % CI, 0.94-0.98), sensitivity 0.92 (95 % CI, 0.88-0.94), pooled specificity 0.91 (95 % CI, 0.87-0.93). The average RQS score of 13 studies using radiomics was 7.8, accounting for 22 % of the total score. The 19 studies using deep learning methods had an average CLAIM score of 20, slightly less than half (48.24 %) the ideal score of 42.00. Conclusions: The AI model for chest imaging could well diagnose COVID-19 and other pneumonias. However, it has not been implemented as a clinical decision-making tool. Future researchers should pay more attention to the quality of research methodology and further improve the generalizability of the developed predictive models.

The Three Two-Pore Channel Subtypes from Rabbit Exhibit Distinct Sensitivity to Phosphoinositides, Voltage, and Extracytosolic pH.

Two pore channels (TPCs) are implicated in vesicle trafficking, virus infection, and autophagy regulation. As Na+- or Ca2+-permeable channels, TPCs have been reported to be activated by NAADP, PI(3,5)P2, and/or high voltage. However, a comparative study on the function and regulation of the three mammalian TPC subtypes is currently lacking. Here, we used the electrophysiological recording of enlarged endolysosome vacuoles, inside-out and outside-out membrane patches to examine the three TPCs of rabbit (Oryctolagus cuniculus, or Oc) heterologously expressed in HEK293 cells. While PI(3,5)P2 evoked Na+ currents with a potency order of OcTPC1 > OcTPC3 > OcTPC2, only OcTPC2 displayed a strict dependence on PI(3,5)P2. Both OcTPC1 and OcTPC3 were activatable by PI3P and OcTPC3 was also activated by additional phosphoinositide species. While OcTPC2 was voltage-independent, OcTPC1 and OcTPC3 showed voltage dependence with OcTPC3 depending on high positive voltages. Finally, while OcTPC2 preferred a luminal pH of 4.6-6.0 in endolysosomes, OcTPC1 was strongly inhibited by extracytosolic pH 5.0 in both voltage-dependent and -independent manners, and OcTPC3 was inhibited by pH 6.0 but potentiated by pH 8.0. Thus, the three OcTPCs form phosphoinositide-activated Na+ channels with different ligand selectivity, voltage dependence, and extracytosolic pH sensitivity, which likely are optimally tuned for function in specific endolysosomal populations.

Emerging viruses: Cross-species transmission of coronaviruses, filoviruses, henipaviruses, and rotaviruses from bats.

Emerging infectious diseases, especially if caused by bat-borne viruses, significantly affect public health and the global economy. There is an urgent need to understand the mechanism of interspecies transmission, particularly to humans. Viral genetics; host factors, including polymorphisms in the receptors; and ecological, environmental, and population dynamics are major parameters to consider. Here, we describe the taxonomy, geographic distribution, and unique traits of bats associated with their importance as virus reservoirs. Then, we summarize the origin, intermediate hosts, and the current understanding of interspecies transmission of Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, Nipah, Hendra, Ebola, Marburg virus, and rotaviruses. Finally, the molecular interactions of viral surface proteins with host cell receptors are examined, and a comparison of these interactions in humans, intermediate hosts, and bats is conducted. This uncovers adaptive mutations in virus spike protein that facilitate cross-species transmission and risk factors associated with the emergence of novel viruses from bats.

Feline Infectious Peritonitis Virus Nsp5 Inhibits Type I Interferon Production by Cleaving NEMO at Multiple Sites

Feline infectious peritonitis (FIP), caused by virulent feline coronavirus, is the leading infectious cause of death in cats. The type I interferon (type I IFN)-mediated immune responses provide host protection from infectious diseases. Several coronaviruses have been reported to evolve diverse strategies to evade host IFN response. However, whether feline infectious peritonitis virus (FIPV) antagonizes the type I IFN signaling remains unclear. In this study, we demonstrated that FIPV strain DF2 infection not only failed to induce interferon-β (IFN-β) and interferon-stimulated gene (ISG) production, but also inhibited Sendai virus (SEV) or polyinosinic-polycytidylic acid (poly(I:C))-induced IFN-β production. Subsequently, we found that one of the non-structural proteins encoded by the FIPV genome, nsp5, interrupted type I IFN signaling in a protease-dependent manner by cleaving the nuclear factor κB (NF-κB) essential modulator (NEMO) at three sites—glutamine132 (Q132), Q205, and Q231. Further investigation revealed that the cleavage products of NEMO lost the ability to activate the IFN-β promoter. Mechanistically, the nsp5-mediated NEMO cleavage disrupted the recruitment of the TRAF family member-associated NF-κB activator (TANK) to NEMO, which reduced the phosphorylation of interferon regulatory factor 3 (IRF3), leading to the inhibition of type I IFN production. Our research provides new insights into the mechanism for FIPV to counteract host innate immune response. [ABSTRACT FROM AUTHOR] Copyright of Viruses (1999-4915) is the property of MDPI Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Management in the paediatric wards facing novel coronavirus infection: a rapid review of guidelines and consensuses.

OBJECTIVES: Relevant guidelines and consensuses for COVID-19 contain recommendations aimed at optimising the management in paediatric wards. The goal of this study was to determine the quality of those recommendations and provide suggestions to hospital managers for the adjustment of existing hospital prevention and control strategies, and also to offer recommendations for further research. DESIGN: A rapid review of the guidelines and consensuses for the management in paediatric wards facing COVID-19. METHODS: PubMed, EMBASE, the Cochrane Library, UpToDate, China National Knowledge Infrastructure, the Wanfang database and relevant websites such as medlive.cn, dxy.cn, the National Health and Health Commission and the China Center for Disease Control and Prevention were systematically searched through late May 2020. The Appraisal of Guidelines for Research and Evaluation II (AGREE II) tool was then used to assess the quality of the selected articles and summarise the relevant evidence concerning management in paediatric wards. RESULTS: A total of 35 articles were included, composed of 3 consensus guidelines, 25 expert consensuses and 7 expert opinions. Of the 35 papers, 24 were from China, 2 from the USA, 1 from Spain, 1 from Brazil, 1 from Saudi Arabia and 6 from multinational cooperative studies. Scores for the six domains of the AGREE II tool (scope and purpose, stakeholder involvement, rigour of development, clarity of presentation, applicability and editorial independence) were 98.57%, 53.57%, 17.92%, 69.62%, 26.96% and 50.35%, respectively. Recommendations for nosocomial infection and control, human resource management as well as management of paediatric patients and their families were summarised. CONCLUSIONS: Due to the outbreak of COVID-19, the quality of rapid guidelines and consensuses for the management in paediatric wards affected by COVID-19 is unsatisfactory. In the future, it will be necessary to develop more high-quality guidelines or consensuses for the management in paediatric wards to deal with nosocomial outbreaks in order to fully prepare for emergency medical and health problems.

Bibliometric analysis of researches on traditional Chinese medicine for coronavirus disease 2019 (COVID-19).

BACKGROUND: The coronavirus disease 2019 (COVID-19) has caused a worldwide pandemic, and traditional Chinese medicine (TCM) has played an important role in response. We aimed to analyze the published literature on TCM for COVID-19, and provide reference for later research. METHODS: This study searched the CBM, CNKI, PubMed, and EMBASE from its establishment to March 11, 2020. VOSviewer 1.6.11 and gCLUTO 2.0 software were used to visually analyze the included studies. RESULTS: A total of 309 studies were included, including 61 journals, 1441 authors, 277 institutions, and 27 provinces. Research collaborations among regions were among those close in geographical distance. The collaborations of institutions and authors were more likely to be restricted to the same region. Among the authors with frequency greater than two (65 authors), only 19 authors had connection with others. More than 70% (358/491) of keywords were only presented once, and 20 keywords were shown more than 10 times. Five research topics were identified: Data mining method based analysis on the medication law of Chinese medicine in prevention and management of COVID-19; exploration of active compounds of Chinese medicine for COVID-19 treatment based on network pharmacology and molecular docking; expert consensus and interpretation of COVID-19 treatment; research on the etiology and pathogenesis of COVID-19; and clinical research of TCM for COVID-19 treatment. CONCLUSION: The research hotspots were scattered, and the collaboration between authors and institutions needed to be further strengthened. To improve the quality and efficiency of research output, the integration of scientific research and resources, as well as scientific collaboration are needed.

[Analysis of traditional Chinese medicine from patent information sharing platform of coronavirus disease 2019 (COVID-19)].

Coronavirus disease 2019(COVID-19) is a newly emerged and highly contagious respiratory disease. Traditional Chinese medicine(TCM) has both systematism theory knowledge and clinical practical value in the prevention and treatment of COVID-19. Therefore, it was particularly important to examine the effect of TCM in the prevention and treatment of COVID-19. The patents of TCM might reflect the latest progression of scientific research. We aimed to provide reference for the prevention and treatment of COVID-19 by extracting and analyzing the TCM patents from the Patent Information Sharing Platform of COVID-19. The antiviral TCM patents were screened and exported from the Patent Information Sharing Platform. VOSviewer 1.6.14 was used to visualize and analyze the network of TCM in these patents. There were total 292 TCM patents, including 52 patents for etiological treatment and 240 patents for symptomatic treatment. Thirty-two provinces and 1 076 inventors were involved, mainly from Beijing, Guangdong and Jiangsu. Overall, there were 356 TCMs, 71 single prescriptions, and 221 compound prescriptions. The patents for treatment of coronavirus mainly focused on the treatment of coronavirus, while the patents for symptomatic treatment mainly focuses on the improvement of respiratory symptoms, such as fever and cough. There were 14 highly frequently used TCMs, including Glycyrrhizae Radix et Rhizoma, Scutellariae Radix, Lonicerae Japonicae Flos, Forsythiae Fructus, Isatidis Radix, Astragali Radix, Menthae Haplocalycis Herba, Gypsum Fibrosum, Houttuyniae Herba, Isatidis Folium, Rhei Radix et Rhizoma, Gardeniae Fructus, Platycodonis Radix, Armeniacae Semen Amarum. The analyzed results of the TCM patents from the patent information sharing platform of COVID-19 were consistent with the Guideline of Diagnosis and Treatment of COVID-19(7th edition), and the combination of TCM in each cluster may also provide future directions for drug compatibility.

Longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of SARS-CoV-2 infected patients.

BACKGROUND: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. METHODS: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. FINDINGS: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)]. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8+ T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8+ T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19. INTERPRETATION: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early identification of severe COVID-19 cases. FUNDING: The National Natural Science Foundation of China, the National Science and Technology Major Project, the Health Commission of Hubei Province, Huazhong University of Science and Technology, and the Medical Faculty of the University of Duisburg-Essen and Stiftung Universitaetsmedizin, Hospital Essen, Germany.

COVID-19: Epidemiology, Evolution, and Cross-Disciplinary Perspectives.

The recent outbreak of COVID-19 in Wuhan turned into a public health emergency of international concern. With no antiviral drugs nor vaccines, and the presence of carriers without obvious symptoms, traditional public health intervention measures are significantly less effective. Here, we report the epidemiological and virological characteristics of the COVID-19 outbreak. Originated in bats, 2019-nCoV/ severe acute respiratory syndrome coronavirus (SARS-CoV)-2 likely experienced adaptive evolution in intermediate hosts before transfer to humans at a concentrated source of transmission. Similarities of receptor sequence binding to 2019-nCoV between humans and animals suggest a low species barrier for transmission of the virus to farm animals. We propose, based on the One Health model, that veterinarians and animal specialists should be involved in a cross-disciplinary collaboration in the fight against this epidemic.