Miao Feng’s research while affiliated with Capital institute of Pediatrics and other places

Background Staphylococcus aureus (S. aureus), a prevalent human pathogen known for its propensity to cause severe infections, has exhibited a growing resistance to antibiotics. Lysine acetylation (Kac) is a dynamic and reversible protein post-translational modification (PTM), played important roles in various physiological functions. Recent studies have shed light on the involvement of Kac modification in bacterial antibiotic resistance. However, the precise relationship between Kac modification and antibiotic resistance in S. aureus remains inadequately comprehended. Methods We compared the differential expression of acetylated proteins between erythromycin-resistant (Ery-R) and erythromycin-susceptible (Ery-S) strains of S. aureus by 4D label-free quantitative proteomics technology. Additionally, we employed motif analysis, functional annotation and PPI network to investigate the acetylome landscape and heterogeneity of S. aureus. Furthermore, polysome profiling experiments were performed to assess the translational status of ribosome. Results 6791 Kac sites were identified on 1808 proteins in S. aureus, among which 1907 sites in 483 proteins were quantified. A total of 548 Kac sites on 316 acetylated proteins were differentially expressed by erythromycin pressure. The differentially acetylated proteins were primarily enriched in ribosome assembly, glycolysis and lysine biosynthesis. Bioinformatic analyses implied that Kac modification of ribosomal proteins may play an important role in erythromycin resistance of S. aureus. Western bolt and polysome profiling experiments indicated that the increased Kac levels of ribosomal proteins in the resistant strain may partially offset the inhibitory effect of erythromycin on ribosome function. Conclusions Our findings confirm that Kac modification is related to erythromycin resistance in S. aureus and emphasize the potential roles of ribosomal proteins. These results expand our current knowledge of antibiotic resistance mechanisms, potentially guiding future research on PTM-mediated antibiotic resistance.

Evidence is emerging on the roles of long noncoding RNAs (lncRNAs) as regulatory factors in a variety of viral infection processes, but the mechanisms underlying their functions in coxsackievirus group B type3 (CVB3)-induced acute viral myocarditis have not been explicitly delineated. We previously demonstrated that CVB3 infection decreases miRNA-21 expression; however, lncRNAs that regulate the miRNA-21-dependent CVB3 disease process have yet to be identified. To evaluate lncRNAs upstream of miRNA-21, differentially expressed lncRNAs in CVB3-infected mouse hearts were identified by microarray analysis and lncRNA/miRNA-21 interactions were predicted bioinformatically. MEG3 was identified as a candidate miRNA-21-interacting lncRNA upregulated in CVB3-infected mouse hearts. MEG3 expression was verified to be upregulated in HeLa cells 48 h post CVB3 infection and to act as a competitive endogenous RNA of miRNA-21. MEG3 knockdown resulted in the upregulation of miRNA-21, which inhibited CVB3 replication by attenuating P38-MAPK signaling in vitro and in vivo. Knockdown of MEG3 expression before CVB3 infection inhibited viral replication in mouse hearts and alleviated cardiac injury, which improved survival. Furthermore, the knockdown of CREB5, which was predicted bioinformatically to function upstream of MEG3, was demonstrated to decrease MEG3 expression and CVB3 viral replication. This study identifies the function of the lncRNA MEG3/miRNA-21/P38 MAPK axis in the process of CVB3 replication, for which CREB5 could serve as an upstream modulator.

Objective: To detect viral load in human cytomegalovirus (HCMV) infection children after hematopoietic stem cell transplant (HSCT) by chip digital PCR (cdPCR). Methods: The plasmid pUC57-UL83 containing the HCMV-UL83 gene and HCMV AD169 strain were used to evaluate the sensitivity of cdPCR. Either HSV-1, HSV-2, VZV, EBV, HHV-6, or HHV-7 was used to evaluate the specificity of HCMV cdPCR. The cdPCR was compared with quantitative PCR (qPCR) by detecting HCMV infection in 125 children's whole blood samples following HSCT. Results: The limit of detection (LOD) of HCMV cdPCR was 103 copies/ml and the qPCR LOD was 297 copies/ml for plasmid pUC57-UL83. The result of HCMV cdPCR was 146 copies/ml for the HCMV AD169 strain, indicating that the sensitivity of cdPCR was higher than that of qPCR. There is no cross-reaction between HCMV cdPCR and other herpes viruses. The incidence of HCMV infection was 30.40% in 125 children following HSCT by cdPCR. The range of the HCMV viral load was from 107 copies/ml to 6600 copies/ml by cdPCR. Conclusions: cdPCR is more sensitive than qPCR for detecting HCMV viral load. Furthermore, the cdPCR could be used to detect the viral load of HCMV infection before or after HSCT in children.

Objective To detect viral load in Human Cytomegalovirus (HCMV) infection children after hematopoietic stem cell transplant (HSCT) by chip digital PCR (cdPCR). Methods The plasmid pUC57-UL83 containing the HCMV UL83 gene and HCMV AD169 strain were used to evaluate the sensitivity of cdPCR. Either HSV-1, HSV-2, VZV, EBV, HHV-6, or HHV-7 was used to evaluate the specificity of HCMV cdPCR. The cdPCR was compared with quantitative PCR (qPCR) by detecting HCMV infection of 125 children whole blood samples following HSCT. Results The limit of detection (LOD) of HCMV cdPCR was 103 copies/ml and the qPCR LOD was 297 copies/ml for plasmid pUC57-UL83. The result of HCMV cdPCR was 146 copies/ml for HCMV AD169 strain, indicating that the sensitivity of cdPCR was higher than that of qPCR. There is no cross-reaction between HCMV cdPCR and other herpes viruses. The incident of HCMV infection was 30.40% in 125 children following HSCT by cdPCR. The range of the HCMV viral load was from 107 copies/ml to 6600 copies/ml by cdPCR. Conclusions cdPCR is more sensitive than qPCR for detecting HCMV viral load. Furthermore, the cdPCR could be used to detect the viral load of HCMV infection before or after HSCT in children.

Objective: To detect the Epstein-Barr virus (EBV) viral load of children after hematopoietic stem cell transplantation (HSCT) using chip digital PCR (cdPCR). Methods: The sensitivity of cdPCR was determined using EBV plasmids and the EBV B95-8 strain. The specificity of EBV cdPCR was evaluated using the EBV B95-8 strain and other herpesviruses (herpes simplex virus 1, herpes simplex virus 2, varicella zoster virus, human cytomegalovirus, human herpesvirus 6, and human herpesvirus 7). From May 2019 to September 2020, 64 serum samples of children following HSCT were collected. EBV infection and the viral load of serum samples were detected by cdPCR. The epidemiological characteristics of EBV infections were analyzed in HSCT patients. Results: The limit of detection of EBV cdPCR was 110 copies/mL, and the limit of detection of EBV quantitative PCR was 327 copies/mL for the pUC57-BALF5 plasmid. The result of EBV cdPCR was up to 121 copies/mL in the EBV B95-8 strain, and both were more sensitive than that of quantitative PCR. Using cdPCR, the incidence of EBV infection was 18.75% in 64 children after HSCT. The minimum EBV viral load was 140 copies/mL, and the maximum viral load was 3,209 copies/mL using cdPCR. The average hospital stay of children with EBV infection (184 ± 91 days) was longer than that of children without EBV infection (125 ± 79 days), P = 0.026. Conclusion: EBV cdPCR had good sensitivity and specificity. The incidence of EBV infection was 18.75% in 64 children after HSCT from May 2019 to September 2020. EBV cdPCR could therefore be a novel method to detect EBV viral load in children after HSCT.

Coxsackievirus B3 (CVB3) can cause viral myocarditis, pancreatitis, and aseptic meningitis. This study aimed to construct an engineered CVB3 harboring three different tissue-specific miRNA targets (CVB3-miR3*T) to decrease the virulence of CVB3 in muscles, pancreas, and brain. CVB3-miR3*T and CVB3-miR-CON (containing three sequences not found in the human genome) were engineered and replicated in HELA cells. A viral plaque assay was used to determine the titers in HELA cells and TE671 cells (high miRNA-206 expression), MIN-6 cells (high miRNA-29a-3p expression), and mouse astrocytes (high miRNA-124-3p expression). We found that engineered CVB3 showed attenuated replication and reduced cytotoxicity, the variability of each type of cell was also increased in the CVB3-miR3*T group. Male BALB/c mice were infected to determine the LD50 and examine heart, pancreas, and brain titers and injury. Viral replication of the engineered viruses was restricted in infected mouse heart, pancreas, and brain, and viral plaques were about 100 fold lower compared with the control group. Mice immunized using CVB3-miR3*T, UV-inactivated CVB3-WT, and CVB3-miR-CON were infected with 100 × LD50 of CVB3-WT to determine neutralization. CVB3-miRT*3-preimmunized mice exhibited complete protection and remained alive after lethal virus infection, while only 5/15 were alive in the UV-inactivated mice, and all 15 mice were dead in the PBS-immunized group. The results demonstrate that miR-206-, miRNA-29a-3p-, and miRNA-124-3p-mediated CVB3 detargeting from the pancreas, heart, and brain might be a highly effective strategy for viral vaccine development.

Background To establish a method for detecting HCMV viral load to guide clinical treatment by chip digital PCR (cdPCR). Methods 5.67×10⁶TCID50/ml of HCMV AD169 was serially diluted to evaluate sensitive of cdPCR, HSV-1, HSV-2, VZV, EBV, HHV-6 and HHV-7 were used to evaluate the specificity of HCMV cdPCR. HCMV infection were analyzed in 110 children leukemia whole blood by RT-qPCR and cdPCR. Results The sensitive of HCMV cdPCR was up to 71 ± 32 copies/ml, which is higher than that of RT-qPCR. HCMV cdPCR did not cross react with other herpesviruses. The cdPCR effectively detected 7 HCMV positive samples, making the laboratory diagnosis rate of HCMV increased by 6.36% (7/110) for children leukemia patients. And the prevalence of HCMV infection increased from 28.18–34.54% in 110 children leukemia patients by cdPCR. Conclusion cdPCR is more sensitive to detect viral load than RT-qPCR. The cdPCR may be used to evaluate relationship between viral load and progression of HCMV infection in patients.

Background: The P38 mitogen-activated protein kinase (MAPK) pathway plays an essential role in CVB3-induced diseases. We previously demonstrated microRNA-21 has potential inhibitory effect on the MAP2K3 which locates upstream of P38 MAPK and was upregulated in mouse hearts upon CVB3 infection. However, the effect and underlying mechanism of miRNA-21 on CVB3 infection remain unclear. Methods: We detected continuous changes of cellular miRNA-21 and P38 MAPK proteins expression profiling post CVB3 infection in vitro within 12 h. P38 MAPK signaling was inhibited by the specific inhibitor, small interfering RNA and miRNA-21 mimic in vitro, CVB3 replication, cell apoptosis rate and proliferation were detected. Viral load in the mice heart, cardiomyocyte apoptosis rate and histological of the heart were also detected in the mice model of viral myocarditis pretreated with miRNA-21-lentivirus. Results: We observed significant upregulation of miRNA-21 expression followed by suppression of the MAP2K3/P38 MAPK signaling in CVB3-infected Hela cells. The inactivation of the MAP2K3/P38 MAPK signaling by P38 MAPK specific inhibitor, small interfering RNA against MAP2K3, or miRNA-21 overexpression significantly inhibited viral progeny release from CVB3-infected cells. Mechanistically, when compared with control miRNA, miRNA-21 showed no effect on capsid protein VP1 expression and viral load within host cells, while significantly reversing CVB3-induced caspase-3 activation and cell apoptosis rate, further promoting proliferation of infected cells, which indicates the inhibitory effect of miRNA-21 on CVB3 progeny release. In the in vivo study, when compared with control miRNA, miRNA-21 pretreatment remarkably inactivated the MAP2K3/P38 MAPK signaling in mice and protected them against CVB3 infection as evidenced by significantly alleviated cell apoptosis rate, reduced viral titers, necrosis in the heart as well as by remarkably prolonged survival time. Conclusions: miRNA-21 were reverse correlated with P38 MAPK activation post CVB3 infection, miRNA-21 overexpression significantly inhibited viral progeny release and decreased myocytes apoptosis rate in vitro and in vivo, suggesting that miRNA-21 may serve as a potential therapeutic agent against CVB3 infection through targeting the MAP2K3/P38 MAPK signaling.

Abstract Background Interleukin (IL)-37 has emerged as a novel anti-inflammatory cytokine that play an immunosuppressive role in regulating inflammatory response. This study aimed to measure IL-37 levels in the plasma and peripheral blood mononuclear cells (PBMCs) of patients with systemic juvenile idiopathic arthritis (sJIA), and to establish the correlation between IL-37 levels and disease activity, laboratory parameters and inflammatory cytokines. Methods The mRNA levels of IL-37 in PBMCs and plasma IL-37 concentrations in 46 sJIA patients and 30 age- and sex-matched healthy controls were measured by real-time polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The correlations between plasma IL-37 levels and disease activity, laboratory parameters and inflammatory cytokines in sJIA were analyzed by Spearman correlation test. PBMCs from the sJIA patients were stimulated with recombinant human IL-37 (rhIL-37) protein, expressions of IL-1β, IL-6, TNF-α and IL-17 were detected by RT-PCR and ELISA. Results Plasma levels of IL-37 and relative IL-37 mRNA expression were significantly elevated in sJIA patients, especially in active sJIA patients, when compared with the healthy controls (P