Lidocaine inhibits influenza a virus replication by up-regulating IFNα4 via TBK1-IRF7 and JNK-AP1 signaling pathways.

Influenza A viruses (IAV), significant respiratory pathogenic agents, cause seasonal epidemics and global pandemics in intra- and interannual cycles. Despite effective therapies targeting viral proteins, the continuous generation of drug-resistant IAV strains is challenging. Therefore, exploring novel host-specific antiviral treatment strategies is urgently needed. Here, we found that lidocaine, widely used for local anesthesia and sedation, significantly inhibited H1N1(PR8) replication in macrophages. Interestingly, its antiviral effect did not depend on the inhibition of voltage-gated sodium channels (VGSC), the main target of lidocaine for anesthesia. Lidocaine significantly upregulated early IFN-I, interferon α4 (IFNα4) mRNA, and protein levels, but not those of early IFNß in mouse RAW 264.7 cell line and human THP-1 derived macrophages. Knocking out IFNα4 by CRISPR-Cas9 partly reversed lidocaine's inhibition of PR8 replication in macrophages. Mechanistically, lidocaine upregulated IFNα4 by activating TANK-binding kinase 1 (TBK1)-IRF7 and JNK-AP1 signaling pathways. These findings indicate that lidocaine has an incredible antiviral potential by enhancing IFN-I signaling in macrophages. In conclusion, our results indicate the potential auxiliary role of lidocaine for anti-influenza A virus therapy and even for anti-SARS-CoV-2 virus therapy, especially in the absence of a specific medicine.

Mindfulness-Based Stress Reduction, Cognitive Behavioral Therapy, and Acupuncture in Chronic Low Back Pain: Protocol for Two Linked Randomized Controlled Trials.

BACKGROUND: Nonpharmacologic mind-body therapies have demonstrated efficacy in low back pain. However, the mechanisms underlying these therapies remain to be fully elucidated. OBJECTIVE: In response to these knowledge gaps, the Stanford Center for Low Back Pain-a collaborative, National Institutes of Health P01-funded, multidisciplinary research center-was established to investigate the common and distinct biobehavioral mechanisms of three mind-body therapies for chronic low back pain: cognitive behavioral therapy (CBT) that is used to treat pain, mindfulness-based stress reduction (MBSR), and electroacupuncture. Here, we describe the design and implementation of the center structure and the associated randomized controlled trials for characterizing the mechanisms of chronic low back pain treatments. METHODS: The multidisciplinary center is running two randomized controlled trials that share common resources for recruitment, enrollment, study execution, and data acquisition. We expect to recruit over 300 chronic low back pain participants across two projects and across different treatment arms within each project. The first project will examine pain-CBT compared with MBSR and a wait-list control group. The second project will examine real versus sham electroacupuncture. We will use behavioral, psychophysical, physical measure, and neuroimaging techniques to characterize the central pain modulatory and emotion regulatory systems in chronic low back pain at baseline and longitudinally. We will characterize how these interventions impact these systems, characterize the longitudinal treatment effects, and identify predictors of treatment efficacy. RESULTS: Participant recruitment began on March 17, 2015, and will end in March 2023. Recruitment was halted in March 2020 due to COVID-19 and resumed in December 2021. CONCLUSIONS: This center uses a comprehensive approach to study chronic low back pain. Findings are expected to significantly advance our understanding in (1) the baseline and longitudinal mechanisms of chronic low back pain, (2) the common and distinctive mechanisms of three mind-body therapies, and (3) predictors of treatment response, thereby informing future delivery of nonpharmacologic chronic low back pain treatments. TRIAL REGISTRATION: ClinicalTrials.gov NCT02503475; https://clinicaltrials.gov/ct2/show/NCT02503475. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): PRR1-10.2196/37823.

Structural insights into Nirmatrelvir (PF-07321332)-3C-like SARS-CoV-2 protease complexation: a ligand Gaussian accelerated molecular dynamics study.

Coronavirus 3C-like protease (3CLpro) is found in SARS-CoV-2 virus, which causes COVID-19. 3CLpro controls virus replication and is a major target for target-based antiviral discovery. As reported by Pfizer, Nirmatrelvir (PF-07321332) is a competitive protein inhibitor and a clinical candidate for orally delivered medication. However, the binding mechanisms between Nirmatrelvir and 3CLpro complex structures remain unknown. This study incorporated ligand Gaussian accelerated molecular dynamics, the one-dimensional and two-dimensional potential of mean force, normal molecular dynamics, and Kramers' rate theory to determine the binding and dissociation rate constants (koff and kon) associated with the binding of the 3CLpro protein to the Nirmatrelvir inhibitor. The proposed approach addresses the challenges in designing small-molecule antiviral drugs.

Towards developing a beef meat export oriented policy in Tanzania: -Exploring the factors that influence beef meat exports.

The purpose of this study was to investigate the factors that influence beef meat exports in Tanzania, with a particular focus on the years 1985 to 2020, in enhancing the development of beef meat export-oriented policy in Tanzania, thereby enhancing beef exports in Tanzania. A time-series panel dataset was analyzed using both descriptive statistics and ordinary least squares (OLS) linear regression analyses models. As per the descriptive analyses, beef meat exports reached the highest pick of 4,300 tons per year in 1990, whereas from 1991 to date, beef meat exportation in Tanzania has been in declining trends despite an increase in beef meat output and trade openness from 162,500 to 486,736 tons and 7.6 to 98.7%, respectively. Nevertheless, while the prospect of Tanzanian beef meat exports appears bright and promising, the industry will continue to encounter trade barriers and must stay competitive to produce enough volume and quality beef meat to meet the needs of its existing and expanding markets. This is because, Tanzanian beef meat competes for market share with beef meat from other countries in the global markets, where customers pay a premium for lines of beef meat that meet quality standards while discarding those that do not. This indicates that the quantity of beef meat produced has no relevance to its world market share, but its quality standards do. Furthermore, the econometric results revealed that the coefficients of the terms of trade, Tanzania GDP per capita, global beef meat consumption, trade openness, and beef meat outputs were found to be significantly positive (P < 0.05) influencing beef meat exports in Tanzania, whereas the trading partners' GDP per capita and exchange rate were not. The findings have varying implications as to what factors need to be addressed to further improve beef meat exports. From the farmer's perspective, better access to adequate funds as a result of increased income benefit from export will assist in improving beef cattle productivity and quality to compete effectively in the global markets. From the government's perspective, because trade openness promotes economic growth through export benefits, the Tanzania government and policymakers need to establish balanced policies to strengthen bilateral trade relationships to generate more opportunities in global markets.

Cytoplasmic Tail Truncation of SARS-CoV-2 Spike Protein Enhances Titer of Pseudotyped Vectors but Masks the Effect of the D614G Mutation.

The high pathogenicity of SARS-CoV-2 requires it to be handled under biosafety level 3 conditions. Consequently, Spike protein-pseudotyped vectors are a useful tool to study viral entry and its inhibition, with retroviral, lentiviral (LV), and vesicular stomatitis virus (VSV) vectors the most commonly used systems. Methods to increase the titer of such vectors commonly include concentration by ultracentrifugation and truncation of the Spike protein cytoplasmic tail. However, limited studies have examined whether such a modification also impacts the protein's function. Here, we optimized concentration methods for SARS-CoV-2 Spike-pseudotyped VSV vectors, finding that tangential flow filtration produced vectors with more consistent titers than ultracentrifugation. We also examined the impact of Spike tail truncation on transduction of various cell types and sensitivity to convalescent serum neutralization. We found that tail truncation increased Spike incorporation into both LV and VSV vectors and resulted in enhanced titers but had no impact on sensitivity to convalescent serum. In addition, we analyzed the effect of the D614G mutation, which became a dominant SARS-CoV-2 variant early in the pandemic. Our studies revealed that, similar to the tail truncation, D614G independently increases Spike incorporation and vector titers, but this effect is masked by also including the cytoplasmic tail truncation. Therefore, the use of full-length Spike protein, combined with tangential flow filtration, is recommended as a method to generate high titer pseudotyped vectors that retain native Spike protein functions. IMPORTANCE Pseudotyped viral vectors are useful tools to study the properties of viral fusion proteins, especially those from highly pathogenic viruses. The Spike protein of SARS-CoV-2 has been investigated using pseudotyped lentiviral and VSV vector systems, where truncation of its cytoplasmic tail is commonly used to enhance Spike incorporation into vectors and to increase the titers of the resulting vectors. However, our studies have shown that such effects can also mask the phenotype of the D614G mutation in the ectodomain of the protein, which was a dominant variant arising early in the COVID-19 pandemic. To better ensure the authenticity of Spike protein phenotypes when using pseudotyped vectors, we recommend using full-length Spike proteins, combined with tangential flow filtration methods of concentration if higher-titer vectors are required.

Single-cell immune profiling reveals distinct immune response in asymptomatic COVID-19 patients.

While some individuals infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) present mild-to-severe disease, many SARS-CoV-2-infected individuals are asymptomatic. We sought to identify the distinction of immune response between asymptomatic and moderate patients. We performed single-cell transcriptome and T-cell/B-cell receptor (TCR/BCR) sequencing in 37 longitudinal collected peripheral blood mononuclear cell samples from asymptomatic, moderate, and severe patients with healthy controls. Asymptomatic patients displayed increased CD56briCD16- natural killer (NK) cells and upregulation of interferon-gamma in effector CD4+ and CD8+ T cells and NK cells. They showed more robust TCR clonal expansion, especially in effector CD4+ T cells, but lack strong BCR clonal expansion compared to moderate patients. Moreover, asymptomatic patients have lower interferon-stimulated genes (ISGs) expression in general but large interpatient variability, whereas moderate patients showed various magnitude and temporal dynamics of the ISGs expression across multiple cell populations but lower than a patient with severe disease. Our data provide evidence of different immune signatures to SARS-CoV-2 in asymptomatic infections.

Polyethylene Glycol Immunogenicity: Theoretical, Clinical, and Practical Aspects of Anti-Polyethylene Glycol Antibodies.

Polyethylene glycol (PEG) is a flexible, hydrophilic simple polymer that is physically attached to peptides, proteins, nucleic acids, liposomes, and nanoparticles to reduce renal clearance, block antibody and protein binding sites, and enhance the half-life and efficacy of therapeutic molecules. Some naïve individuals have pre-existing antibodies that can bind to PEG, and some PEG-modified compounds induce additional antibodies against PEG, which can adversely impact drug efficacy and safety. Here we provide a framework to better understand PEG immunogenicity and how antibodies against PEG affect pegylated drug and nanoparticles. Analysis of published studies reveals rules for predicting accelerated blood clearance of pegylated medicine and therapeutic liposomes. Experimental studies of anti-PEG antibody binding to different forms, sizes, and immobilization states of PEG are also provided. The widespread use of SARS-CoV-2 RNA vaccines that incorporate PEG in lipid nanoparticles make understanding possible effects of anti-PEG antibodies on pegylated medicines even more critical.

Histone deficiency and accelerated replication stress in T cell aging.

With increasing age, individuals are more vulnerable to viral infections such as with influenza or the SARS-CoV-2 virus. One age-associated defect in human T cells is the reduced expression of miR-181a. miR-181ab1 deficiency in peripheral murine T cells causes delayed viral clearance after infection, resembling human immune aging. Here we show that naive T cells from older individuals as well as miR-181ab1-deficient murine T cells develop excessive replication stress after activation, due to reduced histone expression and delayed S-phase cell cycle progression. Reduced histone expression was caused by the miR-181a target SIRT1 that directly repressed transcription of histone genes by binding to their promoters and reducing histone acetylation. Inhibition of SIRT1 activity or SIRT1 silencing increased histone expression, restored cell cycle progression, diminished the replication-stress response, and reduced the production of inflammatory mediators in replicating T cells from old individuals. Correspondingly, treatment with SIRT1 inhibitors improved viral clearance in mice with miR-181a-deficient T cells after LCMV infection. In conclusion, SIRT1 inhibition may be beneficial to treat systemic viral infection in older individuals by targeting antigen-specific T cells that develop replication stress due to miR-181a deficiency.

Survival Analysis of Treatment Efficacy in Comparative Coronavirus Disease 2019 Studies.

For survival analysis in comparative coronavirus disease 2019 trials, the routinely used hazard ratio may not provide a meaningful summary of the treatment effect. The mean survival time difference/ratio is an intuitive, assumption-free alternative. However, for short-term studies, landmark mortality rate differences/ratios are more clinically relevant and should be formally analyzed and reported.

Adjusting Coronavirus Prevalence Estimates for Laboratory Test Kit Error.

Testing representative populations to determine the prevalence or the percentage of the population with active severe acute respiratory syndrome coronavirus 2 infection and/or antibodies to infection is being recommended as essential for making public policy decisions to ease restrictions or to continue enforcing national, state, and local government rules to shelter in place. However, all laboratory tests are imperfect and have estimates of sensitivity and specificity less than 100%-in some cases, considerably less than 100%. That error will lead to biased prevalence estimates. If the true prevalence is low, possibly in the range of 1%-5%, then testing error will lead to a constant background of bias that most likely will be larger, and possibly much larger, than the true prevalence itself. As a result, what is needed is a method for adjusting prevalence estimates for testing error. Methods are outlined in this article for adjusting prevalence estimates for testing error both prospectively in studies being planned and retrospectively in studies that have been conducted. If used, these methods also would help harmonize study results within countries and worldwide. Adjustment can lead to more accurate prevalence estimates and to better policy decisions. However, adjustment will not improve the accuracy of an individual test.

[Role of cyclophilin A during coronavirus replication and the antiviral activities of its inhibitors].

Cyclophilin A (CypA) is a widely distributed and highly conserved protein in organisms. It has peptidyl-prolyl cis/trans isomerase activity and is a receptor for cyclosporin A (CsA). Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses. Seven types of coronaviruses are currently known to infect humans, among which SARS-CoV, MERS-CoV, and SARS-CoV-2 are fatal for humans. It is well established that CypA is essential for the replication of various coronaviruses such as SARS-CoV, CoV-229E, CoV-NL63, and FCoV. Additionally, CsA and its derivatives (ALV, NIM811, etc.) have obvious inhibitory effects on a variety of coronaviruses. These results suggest that CypA is a potential antiviral target and the existing drug CsA might be used as an anti-coronavirus drug. At the end of 2019, SARS-CoV-2 raged in China, which seriously theatern human health and causes huge economic lases. In view of this, we describe the effects of CypA on the replication of coronaviruses and the antiviral activities of its inhibitors, which will provide the scientific basis and ideas for the development of antiviral drugs for SARS-CoV-2.

Risk of pre-term births and major birth defects resulting from paternal intake of COVID-19 medications prior to conception.

OBJECTIVE: With the ongoing COVID-19 pandemic, large numbers of people will receive one of the several medications proposed to treat COVID-19, including patients of reproductive age. Given that some medications have shown adverse effects on sperm quality, there might be a transgenerational concern. We aim at examining the association between drugs proposed to treat COVID-19 when taken by the father around conception and any pre-term birth or major birth defects in offspring in a nation-wide cohort study using Danish registry data. Offspring whose father filled at least one prescription of the following medications in the 3 months preceding conception were considered exposed: chloroquine, hydroxychloroquine, losartan, azithromycin, naproxen, dexamethasone and prednisone. RESULTS: For azithromycin and naproxen, large numbers of offspring were exposed (> 1800 offspring), and we found no association with adverse birth outcomes. For chloroquine, losartan and dexamethasone, exposure was intermediate (~ 900 offspring), and there was no statistically significant association with birth defects. For hydroxychloroquine and prednisone, exposure was limited (< 300 offspring). Our evidence suggests that azithromycin and naproxen are safe with respect to pre-term birth and birth defects. For the other drugs investigated larger exposures are needed for conclusive statements.

Kinetics of SARS-CoV-2 positivity of infected and recovered patients from a single center.

Recurrence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) positive detection in infected but recovered individuals has been reported. Patients who have recovered from coronavirus disease 2019 (COVID-19) could profoundly impact the health care system. We sought to define the kinetics and relevance of PCR-positive recurrence during recovery from acute COVID-19 to better understand risks for prolonged infectivity and reinfection. A series of 414 patients with confirmed SARS-Cov-2 infection, at The Second Affiliated Hospital of Southern University of Science and Technology in Shenzhen, China from January 11 to April 23, 2020. Statistical analyses were performed of the clinical, laboratory, radiologic image, medical treatment, and clinical course of admission/quarantine/readmission data, and a recurrence predictive algorithm was developed. 16.7% recovered patients with PCR positive recurring one to three times, despite being in strict quarantine. Younger patients with mild pulmonary respiratory syndrome had higher risk of PCR positivity recurrence. The recurrence prediction model had an area under the ROC curve of 0.786. This case series provides characteristics of patients with recurrent SARS-CoV-2 positivity. Use of a prediction algorithm may identify patients at high risk of recurrent SARS-CoV-2 positivity and help to establish protocols for health policy.

Selecting appropriate endpoints for assessing treatment effects in comparative clinical studies for COVID-19.

To evaluate the efficacy and safety of a new treatment for COVID-19 vs. standard care, certain key endpoints are related to the duration of a specific event, such as hospitalization, ICU stay, or receipt of supplemental oxygen. However, since patients may die in the hospital during study follow-up, using, for example, the duration of hospitalization to assess treatment efficacy can be misleading. If the treatment tends to prolong patients' survival compared with standard care, patients in the new treatment group may spend more time in hospital. This can lead to a "survival bias" issue, where a treatment that is effective for preventing death appears to prolong an undesirable outcome. On the other hand, by using hospital-free survival time as the endpoint, we can circumvent the survival bias issue. In this article, we use reconstructed data from a recent, large clinical trial for COVID-19 to illustrate the advantages of this approach. For the analysis of ICU stay or oxygen usage, where the initiating event is potentially an outcome of treatment, standard survival analysis techniques may not be appropriate. We also discuss issues with analyzing the durations of such events.

Risk of pre-term births and major birth defects resulting from paternal intake of COVID-19 medications prior to conception.

OBJECTIVE: With the ongoing COVID-19 pandemic, large numbers of people will receive one of the several medications proposed to treat COVID-19, including patients of reproductive age. Given that some medications have shown adverse effects on sperm quality, there might be a transgenerational concern. We aim at examining the association between drugs proposed to treat COVID-19 when taken by the father around conception and any pre-term birth or major birth defects in offspring in a nation-wide cohort study using Danish registry data. Offspring whose father filled at least one prescription of the following medications in the three months preceding conception were considered exposed: chloroquine, hydroxychloroquine, losartan, azithromycin, naproxen, dexamethasone and prednisone. RESULTS: For azithromycin and naproxen, large numbers of offspring were exposed (> 1800 offspring), and we found no association with adverse birth outcomes. For chloroquine, losartan and dexamethasone, exposure was intermediate (~ 900 offspring), and there was no statistically significant association with birth defects. For hydroxychloroquine and prednisone, exposure was limited (< 300 offspring). There is strong evidence that azithromycin and naproxen are safe with respect to pre-term birth and birth defects. With some caution, the other drugs investigated can be considered safe.

Multi-organ Proteomic Landscape of COVID-19 Autopsies (preprint)

The molecular pathology of multi-organ injuries in COVID-19 patients remains unclear, preventing effective therapeutics development. Here, we report an in-depth multi-organ proteomic landscape of COVID-19 patient autopsy samples. By integrative analysis of proteomes of seven organs, namely lung, spleen, liver, heart, kidney, thyroid and testis, we characterized 11,394 proteins, in which 5336 were perturbed in COVID-19 patients compared to controls. Our data showed that CTSL, rather than ACE2, was significantly upregulated in the lung from COVID-19 patients. Dysregulation of protein translation, glucose metabolism, fatty acid metabolism was detected in multiple organs. Our data suggested upon SARS-CoV-2 infection, hyperinflammation might be triggered which in turn induces damage of gas exchange barrier in the lung, leading to hypoxia, angiogenesis, coagulation and fibrosis in the lung, kidney, spleen, liver, heart and thyroid. Evidence for testicular injuries included reduced Leydig cells, suppressed cholesterol biosynthesis and sperm mobility. In summary, this study depicts the multi-organ proteomic landscape of COVID-19 autopsies, and uncovered dysregulated proteins and biological processes, offering novel therapeutic clues. HIGHLIGHTSO_LICharacterization of 5336 regulated proteins out of 11,394 quantified proteins in the lung, spleen, liver, kidney, heart, thyroid and testis autopsies from 19 patients died from COVID-19. C_LIO_LICTSL, rather than ACE2, was significantly upregulated in the lung from COVID-19 patients. C_LIO_LIEvidence for suppression of glucose metabolism in the spleen, liver and kidney; suppression of fatty acid metabolism in the kidney; enhanced fatty acid metabolism in the lung, spleen, liver, heart and thyroid from COVID-19 patients; enhanced protein translation initiation in the lung, liver, renal medulla and thyroid. C_LIO_LITentative model for multi-organ injuries in patients died from COVID-19: SARS-CoV-2 infection triggers hyperinflammatory which in turn induces damage of gas exchange barrier in the lung, leading to hypoxia, angiogenesis, coagulation and fibrosis in the lung, kidney, spleen, liver, heart, kidney and thyroid. C_LIO_LITesticular injuries in COVID-19 patients included reduced Leydig cells, suppressed cholesterol biosynthesis and sperm mobility. C_LI

How to Quantify and Interpret Treatment Effects in Comparative Clinical Studies of COVID-19.

Clinical trials of treatments for coronavirus disease 2019 (COVID-19) draw intense public attention. More than ever, valid, transparent, and intuitive summaries of the treatment effects, including efficacy and harm, are needed. In recently published and ongoing randomized comparative trials evaluating treatments for COVID-19, time to a positive outcome, such as recovery or improvement, has repeatedly been used as either the primary or key secondary end point. Because patients may die before recovery or improvement, data analysis of this end point faces a competing risk problem. Commonly used survival analysis techniques, such as the Kaplan-Meier method, often are not appropriate for such situations. Moreover, almost all trials have quantified treatment effects by using the hazard ratio, which is difficult to interpret for a positive event, especially in the presence of competing risks. Using 2 recent trials evaluating treatments (remdesivir and convalescent plasma) for COVID-19 as examples, a valid, well-established yet underused procedure is presented for estimating the cumulative recovery or improvement rate curve across the study period. Furthermore, an intuitive and clinically interpretable summary of treatment efficacy based on this curve is also proposed. Clinical investigators are encouraged to consider applying these methods for quantifying treatment effects in future studies of COVID-19.

Proteomics Uncovers Immunosuppression in COVID-19 Patients with Long Disease Course (preprint)

Little is known regarding why a subset of COVID-19 patients exhibited prolonged positivity of SARS-CoV-2 infection. Here, we present a longitudinal sera proteomic resource for 37 COVID-19 patients over nine weeks, in which 2700 proteins were quantified with high quality. Remarkably, we found that during the first three weeks since disease onset, while clinical symptoms and outcome were indistinguishable, patients with prolonged disease course displayed characteristic immunological responses including enhanced Natural Killer (NK) cell-mediated innate immunity and regulatory T cell-mediated immunosuppression. We further showed that it is possible to predict the length of disease course using machine learning based on blood protein levels during the first three weeks. Validation in an independent cohort achieved an accuracy of 82%. In summary, this study presents a rich serum proteomic resource to understand host responses in COVID-19 patients and identifies characteristic Treg-mediated immunosuppression in LC patients, nominating new therapeutic target and diagnosis strategy.

Proteomic and Metabolomic Characterization of COVID-19 Patient Sera (preprint)

Severe COVID-19 patients account for most of the mortality of this disease. Early detection and effective treatment of severe patients remain major challenges. Here, we performed proteomic and metabolomic profiling of sera from 46 COVID-19 and 53 control individuals. We then trained a machine learning model using proteomic and metabolomic measurements from a training cohort of 18 non-severe and 13 severe patients. The model correctly classified severe patients with an accuracy of 93.5%, and was further validated using ten independent patients, seven of which were correctly classified. We identified molecular changes in the sera of COVID-19 patients implicating dysregulation of macrophage, platelet degranulation and complement system pathways, and massive metabolic suppression. This study shows that it is possible to predict progression to severe COVID-19 disease using serum protein and metabolite biomarkers. Our data also uncovered molecular pathophysiology of COVID-19 with potential for developing anti-viral therapies.Funding: This work is supported by grants from Westlake Special Program for COVID19 (2020), and Tencent foundation (2020), National Natural Science Foundation of China (81972492, 21904107, 81672086), Zhejiang Provincial Natural Science Foundation for Distinguished Young Scholars (LR19C050001), Hangzhou Agriculture and Society Advancement Program (20190101A04). Conflict of Interest: The research group of T.G. is partly supported by Tencent, Thermo Fisher Scientific, SCIEX and Pressure Biosciences Inc. C.Z., Z.K., Z.K. and S.Q. are employees of DIAN Diagnostics.