Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known ...about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.
Dual-specificity phosphatase 6 (DUSP6) serves a specific and conserved function on the dephosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). We previously identified Dusp6 as a ...regenerative repressor during zebrafish heart regeneration, therefore we propose to investigate the role of this repressor in mammalian cardiac repair. Utilizing a rat strain harboring Dusp6 nonsense mutation, rat neutrophil-cardiomyocyte co-culture, bone marrow transplanted rats and neutrophil-specific Dusp6 knockout mice, we find that Dusp6 deficiency improves cardiac outcomes by predominantly attenuating neutrophil-mediated myocardial damage in acute inflammatory phase after myocardial infarction. Mechanistically, Dusp6 is transcriptionally activated by p38-C/EBPβ signaling and acts as an effector for maintaining p-p38 activity by down-regulating pERK and p38-targeting phosphatases DUSP1/DUSP16. Our findings provide robust animal models and novel insights for neutrophil-mediated cardiac damage and demonstrate the potential of DUSP6 as a therapeutic target for post-MI cardiac remodeling and other relevant inflammatory diseases.
The zebrafish possesses a remarkable capacity of adult heart regeneration, but the underlying mechanisms are not well understood. Here we report that chromatin remodelling factor Brg1 is essential ...for adult heart regeneration. Brg1 mRNA and protein are induced during heart regeneration. Transgenic over-expression of dominant-negative Xenopus Brg1 inhibits the formation of BrdU
/Mef2C
and Tg(gata4:EGFP) cardiomyocytes, leading to severe cardiac fibrosis and compromised myocardial regeneration. RNA-seq and RNAscope analyses reveal that inhibition of Brg1 increases the expression of cyclin-dependent kinase inhibitors such as cdkn1a and cdkn1c in the myocardium after ventricular resection; and accordingly, myocardial-specific expression of dn-xBrg1 blunts myocardial proliferation and regeneration. Mechanistically, injury-induced Brg1, via its interaction with Dnmt3ab, suppresses the expression of cdkn1c by increasing the methylation level of CpG sites at the cdkn1c promoter. Taken together, our results suggest that Brg1 promotes heart regeneration by repressing cyclin-dependent kinase inhibitors partly through Dnmt3ab-dependent DNA methylation.
Mutations of MECP2 (Methyl-CpG Binding Protein 2) cause Rett syndrome. As a chromatin-associated multifunctional protein, how MeCP2 integrates external signals and regulates neuronal function remain ...unclear. Although neuronal activity-induced phosphorylation of MeCP2 at serine 421 (S421) has been reported, the full spectrum of MeCP2 phosphorylation together with the in vivo function of such modifications are yet to be revealed. Here, we report the identification of several MeCP2 phosphorylation sites in normal and epileptic brains from multiple species. We demonstrate that serine 80 (S80) phosphorylation of MeCP2 is critical as its mutation into alanine (S80A) in transgenic knock-in mice leads to locomotor deficits. S80A mutation attenuates MeCP2 chromatin association at several gene promoters in resting neurons and leads to transcription changes of a small number of genes. Calcium influx in neurons causes dephosphorylation at S80, potentially contributing to its dissociation from the chromatin. We postulate that phosphorylation of MeCP2 modulates its dynamic function in neurons transiting between resting and active states within neural circuits that underlie behaviors.
To combat the continued pandemic of COVID-19, multiplex serological assays have been developed to comprehensively monitor the humoral immune response and help to design new vaccination protocols to ...different SARS-CoV-2 variants. However, multiplex beads and stably transfected cell lines require stringent production and storage conditions, and assays based on flow cytometry is time-consuming and its application is therefore restricted. Here, we describe a phage display system to distinguish the differences of immune response to antigenic domains of multiple SARS-CoV-2 variants simultaneously. Compared with linear peptides, the recombinant antigens displayed on the phage surface have shown some function that requires the correct folding to form a stable structure, and the binding efficiency between the recombinant phage and existing antibodies is reduced by mutations on antigens known to be important for antigen–antibody interaction. By using Phage display mediated immuno-multiplex quantitative PCR (Pi-mqPCR), the binding efficiency between the antibody and antigens of different SARS-CoV-2 variants can be measured in one amplification reaction. Overall, these data show that this assay is a valuable tool to evaluate the humoral response to the same antigen of different SARS-CoV-2 variants or antigens of different pathogens. Combined with high-throughput DNA sequencing technology, this phage display system can be further applied in monitoring humoral immune response in a large population before and after vaccination.
Rett syndrome (RTT) is a progressive neurological disorder caused by mutations in the X-linked protein methyl-CpG-binding protein 2 (MeCP2). The endogenous function of MeCP2 during neural ...differentiation is still unclear. Here, we report that mecp2 is required for brain development in zebrafish. Mecp2 was broadly expressed initially in embryos and enriched later in the brain. Either morpholino knockdown or genetic depletion of mecp2 inhibited neuronal differentiation, whereas its overexpression promoted neuronal differentiation, suggesting an essential role of mecp2 in directing neural precursors into differentiated neurons. Mechanistically, her2 (the zebrafish ortholog of mammalian Hes5) was upregulated in mecp2 morphants in an Id1-dependent manner. Moreover, knockdown of either her2 or id1 fully rescued neuronal differentiation in mecp2 morphants. These results suggest that Mecp2 plays an important role in neural cell development by suppressing the Id1-Her2 axis, and provide new evidence that embryonic neural defects contribute to the later motor and cognitive dysfunctions in RTT.
Flavanomarein is the main component of Coreopsis tinctoria Nutt. (C. tinctoria), which is a globally well-known flower tea that has a distinct flavor and many beneficial health effects, such as ...antioxidant activities. We aimed to explore the effect of flavanomarein on a 6-hydroxydopamine (6-OHDA)-lesioned cell model of oxidative stress.
In this study, we used 6-OHDA-lesioned PC12 cells and primary cortical neurons to investigate the protective effects of flavanomarein and its potential mechanism.
The results indicated that pretreatment with flavanomarein (25, 50, or 100 µM for 24 h) significantly increased the cell viability, reduced the lactate dehydrogenase (LDH) release and improved the mitochondrial membrane potential (∆Ψm) and mitochondrial impairment. Additionally, flavanomarein markedly reduced the gene expression of tumor necrosis factor (TNF)-α and protein kinase C ζ (PKC-ζ), the nuclear translocation of p65, and the levels of p-AMPK-α and acetyl-p53. Flavanomarein also elevated the gene expression of P85α, PKC-β1, and Bcl-2, the protein expression of Sirt1 and ICAD, and the phosphorylation level of AKT.
Together, these results suggest that flavanomarein protects PC12 cells and primary cortical neurons from 6-OHDA-induced neurotoxicity by upregulating the PI3K/AKT signaling pathway and attenuating the nuclear factor kappa B (NF-κB) signaling pathway. Therefore, our study provides evidence that may aid in the development of a potential compound against 6-OHDA toxicity.
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An infusion of Coreopsis tinctoria (CT) flowering tops is traditionally used in Portugal to control hyperglycemia; however, the effects of CT protection against high-fat diet (HFD)-induced hepatic ...insulin resistance have not been systematically studied and the precise mechanism of action is not clear. The metabolomic profiles of insulin-resistant rats fed a HFD and a CT-supplemented diet (HFD supplemented with CT drinking) for 8 weeks were investigated. Serum samples for clinical biochemistry and liver samples for histopathology and liquid chromatography-mass spectrometry-based metabolomic research were collected. Western blot and quantitative real-time PCR analyses were further used to measure the expression of several relevant enzymes together with perturbed metabolic pathways. Using analysis software, the CT treatment was found to significantly ameliorate the disturbance in 10 metabolic pathways. Combined metabolomic, Western blot, and quantitative real-time PCR analyses revealed that CT treatment significantly improved the glucose homeostasis by, on the one hand, through inhibiting the expression of gluconeogenic pathway key proteins glucose-6-phosphatase and phosphoenolpyruvate carboxykinase and, on the other hand, via regulating the mRNA or protein levels of the Krebs cycle critical enzymes (citrate synthase, succinate dehydrogenase complex, subunit A, flavoprotein, and dihydrolipoamide S-succinyltransferase). These results provide metabolic evidence of the complex pathogenic mechanism involved in hepatic insulin resistance and that the supplementation with CT improves insulin resistance at a global scale. Liquid chromatography-mass spectrometry-based metabolomics approaches are helpful to further understand diabetes-related mechanisms.
Most neutralizing antibodies neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by directly blocking the interactions between the spike glycoprotein receptor-binding domain ...(RBD) and its receptor, human angiotensin-converting enzyme 2 (ACE2). Here, we report a novel nanobody (Nb) identified by an RBD-ACE2 competitive panning method that could specifically bind to the RBD of SARS-CoV-2 with a high affinity (EC
= 0.03 nM) and successfully block the binding between the RBD and ACE2 recombinant protein. A structural simulation of the RBD-VHH complex also supports a mechanism of the Nb to block the interaction between the RBD and ACE2. A pseudovirus assay of the Nb showed it could neutralize the WT pseudovirus with high potency (IC
= 0.026 μg/mL). Furthermore, we measured its binding to phages displaying RBDs of different SARS-CoV-2 variants and found that it could bind to recombinant phages displaying the RBD of beta and delta variants. This study also provides a method of phage library competitive panning, which could be useful for directly screening high-affinity antibodies targeting important functional regions.
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