Chronological Changes of Viral Shedding in Adult Inpatients With COVID-19 in Wuhan, China.

BACKGROUND: In December 2019, the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) broke out in Wuhan. Epidemiological and clinical characteristics of patients with COVID-19 have been reported, but the relationships between laboratory features and viral load has not been comprehensively described. METHODS: Adult inpatients (≥18 years old) with COVID-19 who underwent multiple (≥5 times) nucleic acid tests with nasal and pharyngeal swabs were recruited from Renmin Hospital of Wuhan University, including general patients (n = 70), severe patients (n = 195), and critical patients (n = 43). Laboratory data, demographic data, and clinical data were extracted from electronic medical records. The fitted polynomial curve was used to explore the association between serial viral loads and illness severity. RESULTS: Viral load of SARS-CoV-2 peaked within the first few days (2-4 days) after admission, then decreased rapidly along with virus rebound under treatment. Critical patients had the highest viral loads, in contrast to the general patients showing the lowest viral loads. The viral loads were higher in sputum compared with nasal and pharyngeal swab (P = .026). The positive rate of respiratory tract samples was significantly higher than that of gastrointestinal tract samples (P < .001). The SARS-CoV-2 viral load was negatively correlated with portion parameters of blood routine and lymphocyte subsets and was positively associated with laboratory features of cardiovascular system. CONCLUSIONS: The serial viral loads of patients revealed whole viral shedding during hospitalization and the resurgence of virus during the treatment, which could be used for early warning of illness severity, thus improve antiviral interventions.

5-Hydroxymethylcytosines in Circulating Cell-Free DNA Reveal Vascular Complications of Type 2 Diabetes.

BACKGROUND: Long-term complications of type 2 diabetes (T2D), such as macrovascular and microvascular events, are the major causes for T2D-related disability and mortality. A clinically convenient, noninvasive approach for monitoring the development of these complications would improve the overall life quality of patients with T2D and help reduce healthcare burden through preventive interventions. METHODS: A selective chemical labeling strategy for 5-hydroxymethylcytosines (5hmC-Seal) was used to profile genome-wide 5hmCs, an emerging class of epigenetic markers implicated in complex diseases including diabetes, in circulating cell-free DNA (cfDNA) from a collection of Chinese patients (n = 62). Differentially modified 5hmC markers between patients with T2D with and without macrovascular/microvascular complications were analyzed under a case-control design. RESULTS: Statistically significant changes in 5hmC markers were associated with T2D-related macrovascular/microvascular complications, involving genes and pathways relevant to vascular biology and diabetes, including insulin resistance and inflammation. A 16-gene 5hmC marker panel accurately distinguished patients with vascular complications from those without [testing set: area under the curve (AUC) = 0.85; 95% CI, 0.73-0.96], outperforming conventional clinical variables such as urinary albumin. In addition, a separate 13-gene 5hmC marker panel could distinguish patients with single complications from those with multiple complications (testing set: AUC = 0.84; 95% CI, 0.68-0.99), showing superiority over conventional clinical variables. CONCLUSIONS: The 5hmC markers in cfDNA reflected the epigenetic changes in patients with T2D who developed macrovascular/microvascular complications. The 5hmC-Seal assay has the potential to be a clinically convenient, noninvasive approach that can be applied in the clinic to monitor the presence and severity of diabetic vascular complications.

Method for the extraction of circulating nucleic acids based on MOF reveals cell-free RNA signatures in liver cancer.

Cell-free RNA (cfRNA) allows assessment of health, status, and phenotype of a variety of human organs and is a potential biomarker to non-invasively diagnose numerous diseases. Nevertheless, there is a lack of highly efficient and bias-free cfRNA isolation technologies due to the low abundance and instability of cfRNA. Here, we developed a reproducible and high-efficiency isolation technology for different types of cell-free nucleic acids (containing cfRNA and viral RNA) in serum/plasma based on the inclusion of nucleic acids by metal-organic framework (MOF) materials, which greatly improved the isolation efficiency and was able to preserve RNA integrity compared with the most widely used research kit method. Importantly, the quality of cfRNA extracted by the MOF method is about 10-fold that of the kit method, and the MOF method isolates more than three times as many different RNA types as the kit method. The whole transcriptome mapping characteristics of cfRNA in serum from patients with liver cancer was described and a cfRNA signature with six cfRNAs was identified to diagnose liver cancer with high diagnostic efficiency (area under curve = 0.905 in the independent validation cohort) using this MOF method. Thus, this new MOF isolation technique will advance the field of liquid biopsy, with the potential to diagnose liver cancer.

The interplay between N6-methyladenosine and precancerous liver disease: molecular functions and mechanisms.

N6-methyladenosine(m6A) is one of the most abundant modifications of mammalian cellular RNAs. m6A regulates various biological functions in epitranscriptomic ways, including RNA stability, decay, splicing, translation and nuclear export. Recent studies have indicated the growing importance of m6A modification in precancerous disease, influencing viral replication, immune escape, and carcinogenesis. Here, we review the role of m6A modification in HBV/HCV infection, NAFLD and liver fibrosis, and its function in liver disease pathogenesis. Our review will provide a new sight for the innovative treatment strategy for precancerous liver disease.

Serum Lipoprotein(a) and High-Density Lipoprotein Cholesterol Associate with Diabetic Nephropathy: Evidence from Machine Learning Perspectives.

Purpose: Diabetic nephropathy (DN) is a common complication of type 2 diabetes mellitus (T2DM) that significantly impacts the quality of life for affected patients. Dyslipidemia is a known risk factor for developing cardiovascular complications in T2DM patients. However, the association between serum lipoprotein(a) (Lp(a)) and high-density lipoprotein cholesterol (HDL-C) with DN requires further investigation. Patients and Methods: For this cross-sectional study, we randomly selected T2DM patients with nephropathy (DN, n = 211) and T2DM patients without nephropathy (T2DM, n = 217) from a cohort of 142,611 patients based on predefined inclusion and exclusion criteria. We collected clinical data from the patients to identify potential risk factors for DN using binary logistic regression and machine learning. After obtaining the feature importance score of clinical indicators by building a random forest classifier, we examined the correlations between Lp(a), HDL-C and the top 10 indicators. Finally, we trained decision tree models with top 10 features using training data and evaluated their performance with independent testing data. Results: Compared to the T2DM group, the DN group had significantly higher serum levels of Lp(a) (p < 0.001) and lower levels of HDL-C (p = 0.028). Lp(a) was identified as a risk factor for DN, while HDL-C was found to be protective. We identified the top 10 indicators that were associated with Lp(a) and/or HDL-C, including urinary albumin (uALB), uALB to creatinine ratio (uACR), cystatin C, creatinine, urinary ɑ1-microglobulin, estimated glomerular filtration rate (eGFR), urinary ß2-microglobulin, urea nitrogen, superoxide dismutase and fibrinogen. The decision tree models trained using the top 10 features and with uALB at a cut-off value of 31.1 mg/L showed an average area under the receiver operating characteristic curve (AUC) of 0.874, with an AUC range of 0.870 to 0.890. Conclusion: Our findings indicate that serum Lp(a) and HDL-C are associated with DN and we have provided a decision tree model with uALB as a predictor for DN.

The diagnostic and prognostic implications of PRKRA expression in HBV-related hepatocellular carcinoma.

BACKGROUND: Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) accounts for more than half of total HCC patients in developing countries. Currently, HBV-related HCC diagnosis and prognosis still lack specific biomarkers. Here, we investigated if PRKRA expression in peripheral blood could be a potential biomarker for the diagnosis/prognosis of HBV-related HCC. METHODS: The expression of PRKRA in HBV-related HCC was firstly analyzed using TCGA and GEO databases. The results were confirmed in a validation cohort including 152 blood samples from 77 healthy controls and 75 HCC patients, 60 of which were infected with HBV. The potential diagnostic and prognostic values of PRKRA were also evaluated by the area under the receiver operator characteristic curve (AUROC) and Kaplan-Meier method, respectively. RESULTS: PRKRA was significantly upregulated in HCC patients, especially in those with HBV infections. In addition, the combination of PRKRA expression in peripheral blood with serum AFP and CEA levels displayed a better diagnostic performance (AUROC = 0.908, 95% CI 0.844-0.972; p < 0.001). Notably, when serum AFP is less than 200 ng/mL, PRKRA expression demonstrated better diagnostic capability. Furthermore, PRKRA expression levels were associated with expression of EIF2AK2 and inflammatory cytokine genes. CONCLUSIONS: Triple combination testing of blood PRKRA expression, serum AFP and CEA levels could be a noninvasive strategy for diagnosis; and the elevation of PRKRA expression could predicate poor prognosis for HBV-related HCC.

Loss of TARBP2 Drives the Progression of Hepatocellular Carcinoma via miR-145-SERPINE1 Axis.

The clinical outcomes of hepatocellular carcinoma (HCC) remain dismal. Elucidating the molecular mechanisms for the progression of aggressive HCC holds the promise for developing novel intervention strategies. The transactivation response element RNA-binding protein (TRBP/TARBP2), a key component of microRNA (miRNA) processing and maturation machinery has been shown to play conflicting roles in tumor development and progression. We sought to investigate the expression of TARBP2 in HCC using well-characterized HCC cell lines, patient-derived tissues and blood samples. Additionally, the potential prognostic and diagnostic value of TARBP2 in HCC were analyzed using Kaplan-Meier plots and ROC curve. Cell counting kit-8 (CCK-8), wound healing and transwell assays examined the ability of TARBP2 to induce cell proliferation, migration, and invasion in HCC cell lines. RNA sequencing was applied to identify the downstream elements of TARBP2. The interaction of potential targets of TARBP2, miR-145 and serpin family E member 1 (SERPINE1), was assessed using luciferase reporter assay. TARBP2 expression was down-regulated in HCC cell lines relative to normal hepatocyte cells, with a similar pattern further confirmed in tissue and blood samples. Notably, the loss of TARBP2 was demonstrated to promote proliferation, migration, and invasion in HCC cell lines. Interestingly, the reduction of TARBP2 was shown to result in the upregulation of SERPINE1, also known as plasminogen activator inhibitor (PAI-1), which is a vital gene of the HIF-1 signaling pathway. Knockdown of SERPINE1 rescued the TARBP2-lost phenotype. Moreover, TARBP2 depletion induced the upregulation of SERPINE1 through reducing the processing of miR-145, which directly targets SERPINE1. Finally, overexpression of miR-145 repressed SERPINE1 and rescued the functions in sh-TARBP2 HCC cells. Our findings underscore a linear TARBP2-miR-145-SERPINE1 pathway that drives HCC progression, with the potential as a novel intervention target for aggressive HCC.

Transient Early Fine Motor Abnormalities in Infants Born to COVID-19 Mothers Are Associated With Placental Hypoxia and Ischemia.

Background: Long-term effects of Coronavirus Disease 2019 (COVID-19) on infants born to infected mothers are not clear. Fine motor skills are crucial for the development of infant emotional regulation, learning ability and social skills. Methods: Clinical information of 100 infants born to 98 mothers (COVID-19 n = 31, non-COVID-19 n = 67) were collected. Infants were follow-up up to 9 months post-partum. The placental tissues were examined for SARS-CoV-2 infection, pathological changes, cytokines, and mtDNA content. Results: Decreased placental oxygen and nutrient transport capacity were found in infected pregnant women. Increased IL-2, IL-6, TNF-α, and IFN-γ were detected in trophoblast cells and maternal blood of COVID-19 placentas. Elevated early fine motor abnormal-ities and increased serum TNI (troponin I) levels at delivery were observed in infants born to mothers with COVID-19. Increased abnormal mitochondria and elevated mtDNA content were found in the placentas from infected mothers. The placental mtDNA content of three infants with abnormal DDST were increased by 4, 7, and 10%, respectively, compared to the mean of the COVID-19 group. The Maternal Vascular Malperfusion (MVM), elevated cytokines and increased placental mtDNA content in mothers with COVID-19 might be associated with transient early fine motor abnormalities in infants. These abnormalities are only temporary, and they could be corrected by daily training. Conclusions: Babies born to COVID-19 mothers with mild symptoms appeared to have little or no excess long-term risks of abnormal physical and neurobehavioral development as compared with the infants delivered by non-COVID-19 mothers.

A longitudinal sampling study of transcriptomic and epigenetic profiles in patients with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is a novel tick-borne infectious disease caused by a new type of SFTS virus (SFTSV). Here, a longitudinal sampling study is conducted to explore the differences in transcript levels after SFTSV infection, and to characterize the transcriptomic and epigenetic profiles of hospitalized patients. The results reveal significant changes in the mRNA expression of certain genes from onset to recovery. Moreover, m6A-seq reveals that certain genes related with immune regulation may be regulated by m6A. Besides the routine tests such as platelet counts, serum ALT and AST levels testing, distinct changes in myocardial enzymes, coagulation function, and inflammation are well correlated with the clinical data and sequencing data, suggesting that clinical practitioners should monitor the above indicators to track disease progression and guide personalized treatment. In this study, the transcript changes and RNA modification may lend a fresh perspective to our understanding of the SFTSV and play a significant role in the discovery of drugs for effective treatment of this disease.

Corrigendum: Loss of TARBP2 Drives the Progression of Hepatocellular Carcinoma via miR-145-SERPINE1 Axis.

[This corrects the article DOI: 10.3389/fonc.2021.620912.].

TRADES: Targeted RNA Demethylation by SunTag System.

N6-methyladenosine (m6A) is rapidly being studied and uncovered to play a significant role in various biological processes as well as in RNA fate and functions, while the effects of defined m6A sites are rarely characterized for the lack of convenient tools. To provide an applicable method to remove m6A modification at specific loci, an m6A editing system called "targeted RNA demethylation by SunTag system (TRADES)" is engineered. In this system, the targeting element dCas13b is fused to ten copies of GCN4 peptides which can recruit multiple scFv-fusion RNA demethylase to demethylate the target m6A site. Owing to this design, TRADES is more flexible to the indistinct m6A sites for its wide editing window. By site-specific demethylation of messenger RNA (mRNA) SON A2699, the lifetime of SON RNA is successfully prolonged in HeLa cells. Meanwhile, TRADES negligibly influences the lifetime of other non-targeted transcripts. TRADES also can regulate the gene expression of target transcript in an m6A-dependent manner. Moreover, the interference occuring for HBV and HIV replications demonstrates that the TRADES system holds potential in viral life cycle regulation and clinical applications.