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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. SARS-CoV-2 has spread globally. Here, the authors characterize the entry pathway of SARS-CoV-2, show that the SARS-CoV-2 spike protein is less stable than that of SARS-CoV, and show limited cross-neutralization activities between SARS-CoV and SARS-CoV-2 sera.

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. Dusp6, an ERK specific phosphatase, was identified as a regenerative repressor during zebrafish heart regeneration. Here, the authors show that Dusp6 deficiency improves post infarctional cardiac repair by predominantly attenuating neutrophil-mediated myocardial damage in mammalian hearts.

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. The adult zebrafish heart is capable of regeneration but the molecular mechanisms are poorly understood. Here the authors show that chromatin remodeling factor Brg1 represses cyclin-dependent kinase inhibitors to promote myocardial regeneration.

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.

Cas12 and Cas13 are extensively utilized in molecular diagnostics for their trans -cleavage activities, yet their activation characteristics remain partially understood. Here, we conduct an in-depth investigation of Cas12a, Cas12f1, and Cas13a, uncovering the characteristics of their trans -DNase and trans -RNase activities with noncanonical activators. Our findings reveal that DNA can serve as a direct target for CRISPR-Cas13a, markedly increasing the detection sensitivity for single-base mismatches. Moreover, the trans -cleavage activities of Cas12a and Cas13a can be activated by diverse RNA:DNA and RNA:RNA duplexes, respectively, indicating that the presence of stem–loop structures in crRNAs is not essential for their activation. Notably, Cas12f1, unlike Cas12a, exhibits intrinsic RNase activity independently of activation. Leveraging these insights, we have improved the accuracy of a dual-gene target detection approach that employs the CRISPR-Cas12f1 and Cas13a systems. Our research advances the understanding of the noncanonical activation characteristics of Cas12 and Cas13a, contributing to the field of CRISPR-based diagnostics. Uncovering noncanonical trans -cleavage characteristics of Cas12 and Cas13a improves the accuracy of CRISPR-based dual-gene diagnostics and enhances detection sensitivity for single base mismatches through newly discovered activation 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 (EC50 = 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 (IC50 = 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.

Most available neutralizing antibodies are ineffective against highly mutated SARS-CoV-2 Omicron subvariants. Therefore, it is crucial to develop potent and broad-spectrum alternatives to effectively manage Omicron subvariants. Here, we constructed a high-diversity nanobody phage display library and identified nine nanobodies specific to the SARS-CoV-2 receptor-binding domain (RBD). Five of them exhibited cross-neutralization activity against the SARS-CoV-2 wild-type (WT) strain and the Omicron subvariants BA.1 and BA.4/5, and one nanobody demonstrated marked efficacy even against the Omicron subvariants BQ.1.1 and XBB.1. To enhance the therapeutic potential, we engineered a panel of multivalent nanobodies with increased neutralizing potency and breadth. The most potent multivalent nanobody, B13-B13-B13, cross-neutralized all tested pseudoviruses, with a geometric mean of the 50% inhibitory concentration (GM IC50) value of 20.83 ng/mL. An analysis of the mechanism underlying the enhancement of neutralization breadth by representative multivalent nanobodies demonstrated that the strategic engineering approach of combining two or three nanobodies into a multivalent molecule could improve the affinity between a single nanobody and spike, and could enhance tolerance toward escape mutations such as R346T and N460K. Our engineered multivalent nanobodies may be promising drug candidates for treating and preventing infection with Omicron subvariants and even future variants.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-021-22614-1.

MicroRNAs (miRNAs) are critical for both development and function of the central nervous system. Significant evidence suggests that abnormal expression of miRNAs is associated with neurodevelopmental disorders. MeCP2 protein is an epigenetic regulator repressing or activating gene transcription by binding to methylated DNA. Both loss-of-function and gain-of-function mutations in the MECP2 gene lead to neurodevelopmental disorders such as Rett syndrome, autism and MECP2 duplication syndrome. In this study, we demonstrate that miR-130a inhibits neurite outgrowth and reduces dendritic spine density as well as dendritic complexity. Bioinformatics analyses, cell cultures and biochemical experiments indicate that miR-130a targets MECP2 and down-regulates MeCP2 protein expression. Furthermore, expression of the wild-type MeCP2, but not a lossof-function mutant, rescues the miR-130a-induced phenotype. Our study uncovers the MECP2 gene as a previous unknown target for miR-130a, supporting that miR-130a may play a role in neurodevelopment by regulating MeCP2. Together with data from other groups, our work suggests that a feedback regulatory mechanism involving both miR-130a and MeCP2 may serve to ensure their appropriate expression and function in neural development.

Dihydromyricetin (DMY), an important flavanone found in Ampelopsis grossedentata, possesses antioxidative properties that ameliorate skeletal muscle insulin sensitivity and exert a hepatoprotective effect. However, little is known about the effects of DMY in the context of high-fat diet (HFD)-induced hepatic insulin resistance. Male Sprague-Dawley(SD) rats were fed a HFD(60% fat) supplemented with DMY for 8 weeks. The administration of DMY to the rats with HFD-induced insulin resistance reduces hyperglycemia, plasma levels of insulin, and steatosis in the liver. Furthermore, DMY treatment modulated 24 metabolic pathways, including glucose metabolism, the TCA cycle. DMY significantly enhanced glucose uptake and improved the translocation of glucose transporter 1. The specificity of DMY promoted the phosphorylation of AMP-activated protein kinase (AMPK). In addition, the exposure of HepG2 cells to high glucose concentrations impaired the insulin-stimulated phosphorylation of Akt2 Ser474 and insulin receptor substrate-1 (IRS-1) Ser612, increased GSK-3β phosphorylation, and upregulated G6Pase and PEPCK expression. Collectively, DMY improved glucose-related metabolism while reducing lipid levels in the HFD-fed rats. These data suggest that DMY might be a useful drug for use in type 2 diabetes insulin resistance therapy and for the treatment of hepatic steatosis.