Towards Point of Care CRISPR-Based Diagnostics: From Method to Device.

Rapid, accurate, and portable on-site detection is critical in the face of public health emergencies. Infectious disease control and public health emergency policymaking can both be aided by effective and trustworthy point of care tests (POCT). A very promising POCT method appears to be the clustered regularly interspaced short palindromic repeats and associated protein (CRISPR/Cas)-based molecular diagnosis. For on-site detection, CRISPR/Cas-based detection can be combined with multiple signal sensing methods and integrated into smart devices. In this review, sensing methods for CRISPR/Cas-based diagnostics are introduced and the advanced strategies and recent advances in CRISPR/Cas-based POCT are reviewed. Finally, the future perspectives of CRISPR and POCT are summarized and prospected.

How Agricultural Farmers Respond to Risks during the COVID-19 Pandemic: An Exploration through the Dual Social Capitals Approach

The degree of risk to which agricultural farmers are exposed during the COVID-19 pandemic and how they tackle those difficulties is a critical topic. Although the topic has been paid considerable attention by worldwide scholars, this study intends to compensate for it via conducting a ground-breaking analysis based on sample survey data. Integrating theoretical perspectives of individual- and collective-level social capitals rooted in sociology, and using NPRM (Nested Poisson Regression Model) to analyze a sample survey data collected in rural China in August 2020, we generated the following findings. (1) The overall risks and damages to agricultural production and management are relatively minimal. Thus, farmers are highly confident in conquering the pandemic and recovering their business. (2) Compared with micro- and macro-level influencing factors, social capital at both levels could greatly help agricultural farmers obtain informal and formal supporting resources (such as encouragement and financial supports), thus helping them to cope with the pandemic shock. (3) Specifically, the acquisition of informal supporting resources is mainly affected by the size of farmers' ordinary networks (Spring Festival Visiting Network) and the frequency of public activities held in a village;gaining access to formal supporting resources is also influenced by the frequency of public activities, but the state of farmers' personal connections with official departments plays a crucial role in determining the amount of such resources can be obtained. According to these empirical findings, suggestions on how to suppress the negative effects and lift the positive effects of dual social capitals in the process of responding to risks are proposed.

Gaseous and Particulate Pollution in the Wu-Chang-Shi Urban ,Agglomeration on the Northern Slope of Tianshan Mountains from 2017 to 2021

Rapid social development has led to serious air pollution problems in cities, and air pollutants, including gaseous pollutants and particulate matter, have an important impact on climate, the environment, and human health. This study analyzed the characteristics, potential sources, and causes of air pollution in the Wu-Chang-Shi urban cluster. The results showed that NO2, CO, SO2, PM10, and PM2.5 had a tendency to decrease, while O3 showed an increasing trend. The concentrations of SO2, NO2, CO, PM2.5, and PM10 showed the highest values in winter and the lowest values in summer, with similar seasonal variations. However, the concentration of O3 was highest in the summer and lowest in the winter. Compared with the pollutant concentrations in other Chinese cities, PM2.5, PM10, and NO2 are more polluted in the Wu-Chang-Shi urban. Meteorological factors have a greater impact on pollutant concentrations, with higher concentrations of major pollutants observed when wind speeds are low and specific wind directions are observed, and higher secondary pollutant O3 concentrations observed when wind speeds are low and specific wind directions are observed. The backward trajectory and concentration weighting analysis show that the particulate pollutants in the Wu-Chang-Shi urban in winter mainly come from Central Asia and surrounding cities. O3 showed an increasing trend before and after the novel coronavirus outbreak, which may be related to changes in NOX, volatile organic compounds, and solar radiation intensity, and the concentrations of SO2, NO2, CO, PM10, and PM2.5 showed an overall decreasing trend after the outbreak and was smaller than before the outbreak, which is related to the reduction of industrial and anthropogenic source emissions during the outbreak.

Nucleic acid amplification with specific signal filtration and magnification for ultrasensitive colorimetric detection.

Recently, sensitive, fast and low cost nucleic acid isothermal amplification technologies (such as loop-mediated isothermal amplification, LAMP) have attracted great attention in the urgent needs of point-of-care testing (POCT) and regular epidemic prevention and control. However, unlike PCR which usually employs TaqMan probe to report specific signals, specific-signal-output strategies in isothermal amplification are immature and visual detection even rare, which limits their popularity in POCT. We hypothesize to address this issue by designing a visual-signal-report system to both filtrate and magnify the target information in isothermal amplification. In this work, we developed a specific signal filtration and magnification colorimetric isothermal sensing platform (SFMC for short) for ultrasensitive detection of DNA and RNA. SFMC consists of two processes: an isothermal amplification with specific signal filtration and a self-replication catalyzed hairpin assembly (SRCHA) for rapid target-specific signal magnification and outputting. With these unique properties, this biosensing platform could detect target DNA as low as 5 copies per reaction and target RNA as low as 10 copies per reaction by naked eyes. Benefited from the excellent colorimetric detection performance, this biosensing platform has been successfully used for African swine fever virus (ASFV) and SARS-CoV-2 detection.

Plasma metabolome and cytokine profile reveal glycylproline modulating antibody fading in convalescent COVID-19 patients.

The COVID-19 pandemic has incurred tremendous costs worldwide and is still threatening public health in the "new normal." The association between neutralizing antibody levels and metabolic alterations in convalescent patients with COVID-19 is still poorly understood. In the present work, we conducted absolutely quantitative profiling to compare the plasma cytokines and metabolome of ordinary convalescent patients with antibodies (CA), convalescents with rapidly faded antibodies (CO), and healthy subjects. As a result, we identified that cytokines such as M-CSF and IL-12p40 and plasma metabolites such as glycylproline (gly-pro) and long-chain acylcarnitines could be associated with antibody fading in COVID-19 convalescent patients. Following feature selection, we built machine-learning-based classification models using 17 features (six cytokines and 11 metabolites). Overall accuracies of more than 90% were attained in at least six machine-learning models. Of note, the dipeptide gly-pro, a product of enzymatic peptide cleavage catalyzed by dipeptidyl peptidase 4 (DPP4), strongly accumulated in CO individuals compared with the CA group. Furthermore, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination experiments in healthy mice demonstrated that supplementation of gly-pro down-regulates SARS-CoV-2-specific receptor-binding domain antibody levels and suppresses immune responses, whereas the DPP4 inhibitor sitagliptin can counteract the inhibitory effects of gly-pro upon SARS-CoV-2 vaccination. Our findings not only reveal the important role of gly-pro in the immune responses to SARS-CoV-2 infection but also indicate a possible mechanism underlying the beneficial outcomes of treatment with DPP4 inhibitors in convalescent COVID-19 patients, shedding light on therapeutic and vaccination strategies against COVID-19.

Impact of the COVID-19 pandemic on congenital diaphragmatic hernia patients: a single-center retrospective study.

PURPOSE: To investigate the impact of COVID-19 on the treatment of children with congenital diaphragmatic hernia (CDH). METHODS: We retrospectively collected and compared the data of patients with CDH admitted between January 1, 2020 and December 31, 2021(study group) with the CDH patients admitted before the pandemic between January 1, 2018 and December 31, 2019 (control group). RESULTS: During the pandemic, 41 patients with CDH diagnosed prenatally were transferred to our hospital, and 40 underwent surgical repair. The number of patients treated in our hospital increased by 24.2% compared with the 33 patients before the pandemic. During the pandemic, the overall survival rate, postoperative survival rate and recurrence rate were 85.4%, 87.5% and 7.3%, respectively, and there were no significant differences compared with the control group (75.8%, 83.3% and 9.1%, respectively). The average length of hospital stay in patients admitted during the pandemic was longer than that in the control group (31 days vs. 16 days, P < 0.001), and the incidence of nosocomial infection was higher than that in the control group (19.5% vs. 3%, P = 0.037). CONCLUSIONS: CDH patients confirmed to be SARS-CoV-2 infection-free can receive routine treatment. Our data indicate that the implementation of protective measures during the COVID-19 pandemic, along with appropriate screening and case evaluation, do not have a negative impact on the prognosis of children.

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.

Moderated Online Data-Collection for Developmental Research: Methods and Replications.

Online data collection methods are expanding the ease and access of developmental research for researchers and participants alike. While its popularity among developmental scientists has soared during the COVID-19 pandemic, its potential goes beyond just a means for safe, socially distanced data collection. In particular, advances in video conferencing software has enabled researchers to engage in face-to-face interactions with participants from nearly any location at any time. Due to the novelty of these methods, however, many researchers still remain uncertain about the differences in available approaches as well as the validity of online methods more broadly. In this article, we aim to address both issues with a focus on moderated (synchronous) data collected using video-conferencing software (e.g., Zoom). First, we review existing approaches for designing and executing moderated online studies with young children. We also present concrete examples of studies that implemented choice and verbal measures (Studies 1 and 2) and looking time (Studies 3 and 4) across both in-person and online moderated data collection methods. Direct comparison of the two methods within each study as well as a meta-analysis of all studies suggest that the results from the two methods are comparable, providing empirical support for the validity of moderated online data collection. Finally, we discuss current limitations of online data collection and possible solutions, as well as its potential to increase the accessibility, diversity, and replicability of developmental science.

Reviving chloroquine for anti-SARS-CoV-2 treatment with cucurbit[7]uril-based supramolecular formulation.

The wide-spreading SARS-CoV-2 virus has put the world into boiling water for more than a year, however pharmacological therapies to act effectively against coronavirus disease 2019 (COVID-19) remain elusive. Chloroquine (CQ), an antimalarial drug, was found to exhibit promising antiviral activity in vitro and in vivo at a high dosage, thus CQ was approved by the FDA for the emergency use authorization (EUA) in the fight against COVID-19 in the US, but later was revoked the EUA status due to the severe clinical toxicity. Herein, we show that supramolecular formulation of CQ by a macrocyclic host, curcurbit[7]uril (CB[7]), reduced its non-specific toxicity and improved its antiviral activity against coronavirus, working in synergy with CB[7]. CB[7] was found to form 1:1 host-guest complexes with CQ, with a binding constant of ∼104 L/mol. The CQ-CB[7] formulation decreased the cytotoxicity of CQ against Vero E6 and L-02 cell lines. In particular, the cytotoxicity of CQ (60 µmol/L) against both Vero E6 cell line and L-02 cell lines was completely inhibited in the presence of 300 µmol/L and 600 µmol/L CB[7], respectively. Furthermore, the CB[7] alone showed astonishing antiviral activity in SARS-CoV-2 infected Vero E6 cells and mouse hepatitis virus strain A59 (MHV-A59) infected N2A cells, and synergistically improved the antiviral activity of CQ-CB[7], suggesting that CB[7]-based CQ formulation has a great potential as a safe and effective antiviral agent against SARS-CoV-2 and other coronavirus.

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.

Can Clinical Symptoms and Laboratory Results Predict CT Abnormality? Initial Findings Using Novel Machine Learning Techniques in Children With COVID-19 Infections.

The rapid spread of coronavirus 2019 disease (COVID-19) has manifested a global public health crisis, and chest CT has been proven to be a powerful tool for screening, triage, evaluation and prognosis in COVID-19 patients. However, CT is not only costly but also associated with an increased incidence of cancer, in particular for children. This study will question whether clinical symptoms and laboratory results can predict the CT outcomes for the pediatric patients with positive RT-PCR testing results in order to determine the necessity of CT for such a vulnerable group. Clinical data were collected from 244 consecutive pediatric patients (16 years of age and under) treated at Wuhan Children's Hospital with positive RT-PCR testing, and the chest CT were performed within 3 days of clinical data collection, from January 21 to March 8, 2020. This study was approved by the local ethics committee of Wuhan Children's Hospital. Advanced decision tree based machine learning models were developed for the prediction of CT outcomes. Results have shown that age, lymphocyte, neutrophils, ferritin and C-reactive protein are the most related clinical indicators for predicting CT outcomes for pediatric patients with positive RT-PCR testing. Our decision support system has managed to achieve an AUC of 0.84 with 0.82 accuracy and 0.84 sensitivity for predicting CT outcomes. Our model can effectively predict CT outcomes, and our findings have indicated that the use of CT should be reconsidered for pediatric patients, as it may not be indispensable.

Cellular Immune Response to COVID-19 and Potential Immune Modulators.

Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Droplets and contacts serve as the main route of transmission of SARS-CoV-2. The characteristic of the disease is rather heterogeneous, ranging from no symptoms to critical illness. The factors associated with the outcome of COVID-19 have not been completely characterized to date. Inspired by previous studies on the relevance of infectious diseases, viral and host factors related to clinical outcomes have been identified. The severity of COVID-19 is mainly related to host factors, especially cellular immune responses in patients. Patients with mild COVID-19 and improved patients with severe COVID-19 exhibit a normal immune response to effectively eliminate the virus. The immune response in patients with fatal severe COVID-19 includes three stages: normal or hypofunction, hyperactivation, and anergy. Eventually, the patients were unable to resist viral infection and died. Based on our understanding of the kinetics of immune responses during COVID-19, we suggest that type I interferon (IFN) could be administered to patients with severe COVID-19 in the hypofunctional stage, intravenous immunoglobulin (IVIG) and glucocorticoid therapy could be administered in the immune hyperactivation stage. In addition, low molecular weight heparin (LMWH) anticoagulation therapy and anti-infective therapy with antibiotics are recommended in the hyperactivation stage.

Viral dynamics and antibody responses in people with asymptomatic SARS-CoV-2 infection.

Over 40% of the coronavirus disease 2019 (COVID-19) COVID-19 patients were asymptomatically infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the immune responses of these asymptomatic individuals is a critical factor for developing the strategy to contain the COVID-19 pandemic. Here, we determined the viral dynamics and antibody responses among 143 asymptomatic individuals identified in a massive screening of more than 5 million people in eight districts of Wuhan in May 2020. Asymptomatic individuals were admitted to the government-designated centralized sites in accordance with policy. The incidence rate of asymptomatic infection is ~2.92/100,000. These individuals had low viral copy numbers (peaked at 315 copies/mL) and short-lived antibody responses with the estimated diminish time of 69 days. The antibody responses in individuals with persistent SARS-CoV-2 infection is much longer with the estimated diminish time of 257 days. These results imply that the immune responses in the asymptomatic individuals are not potent enough for preventing SARS-CoV-2 re-infection, which has recently been reported in recovered COVID-19 patients. This casts doubt on the efficacy of forming "herd-immunity" through natural SARS-CoV-2 infection and urges for the development of safe and effective vaccines.