Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, we describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin-streptavidin approach to localize repair templates to target sites, we achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. We targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. We also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. We suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.

Purpose: Biomarkers can facilitate diagnosis, monitor treatment response, and assess prognosis in some patients with cancer. YKL-40 and matrix metalloproteinase-9 (MMP-9) are two proteins highly differentially expressed by malignant gliomas.We obtained prospective longitudinal serum samples from patients with gliomas to determine whether YKL-40 or MMP-9 could be used as serum markers. Experimental Design: Serum samples were obtained concurrently with magnetic resonance imaging scans. YKL-40 and MMP-9 were determined by ELISA and the values correlated with the patient's radiographic status and survival. Results: High-grade glioma patients who underwent a surgical resection of their tumor had transient increase of bothYKL-40 and MMP-9 serum levels in the postoperative period. Glioblastoma multiforme (GBM) patients with no radiographic evidence of disease (n = 10 patients, 50 samples) had a significantly lower level ofYKL-40 and MMP-9 than patients with active tumor (n = 66 patients, 209 samples; P = 0.0003 and 0.0002, respectively). Anaplastic glioma patients with no radiographic evidence of disease (n = 32 patients, 107 samples) also had a significantly lower level of YKL-40 compared with those patients with active tumor (n = 48 patients, 199 samples; P = 0.04). There was a significant inverse association between YKL-40 and survival in GBM, hazard ratio (hazard ratio, 1.4; P = 0.02), and anaplastic astrocytoma patients (hazard ratio, 2.2; P = 0.05). Conclusions: YKL-40 and MMP-9 can be monitored in patients' serum and help confirm the absence of active disease in GBM and YKL-40 in anaplastic glioma patients. YKL-40 can be used as predictor of survival in patients with high-grade glioma. Longitudinal studies with a larger patient population are needed to confirm these findings.

Background Colistin resistance mediated by mcr-1 -harbouring plasmids is an emerging threat in Enterobacteriaceae, like Salmonella . Based on its major contribution to the diarrhoea burden, the epidemic state and threat of mcr-1 -harbouring Salmonella in community-acquired infections should be estimated. Methods This retrospective study analysed the mcr-1 gene incidence in Salmonella strains collected from a surveillance on diarrhoeal outpatients in Shanghai Municipality, China, 2006–2016. Molecular characteristics of the mcr-1 -positive strains and their plasmids were determined by genome sequencing. The transfer abilities of these plasmids were measured with various conjugation strains, species, and serotypes. Findings Among the 12,053 Salmonella isolates, 37 mcr-1 -harbouring strains, in which 35 were serovar Typhimurium, were detected first in 2012 and with increasing frequency after 2015. Most patients infected with mcr-1 -harbouring strains were aged <5 years. All strains, including fluoroquinolone-resistant and/or extended-spectrum β-lactamase-producing strains, were multi-drug resistant. S. Typhimurium had higher mcr-1 plasmid acquisition ability compared with other common serovars. Phylogeny based on the genomes combined with complete plasmid sequences revealed some clusters, suggesting the presence of mcr-1 -harbouring Salmonella outbreaks in the community. Most mcr-1 -positive strains were clustered together with the pork strains, strongly suggesting pork consumption as a main infection source. Interpretation The mcr-1 -harbouring Salmonella prevalence in community-acquired diarrhoea displays a rapid increase trend, and the ESBL -mcr-1 -harbouring Salmonella poses a threat for children. These findings highlight the necessary and significance of prohibiting colistin use in animals and continuous monitoring of mcr-1 -harbouring Salmonella .

A number of circular RNAs (circRNAs) have been implicated in rheumatoid arthritis (RA) pathogenesis; however, little is known about their function and hidden molecular mechanism in immune and inflammation regulation. We investigated the role and the underlying mechanism of circRNA_09505 in RA in this study. Real-time PCR and fluorescence in situ hybridization (FISH) are adopted to estimate the quantitative expression and localization of circRNA_09505 in macrophages. The altering effect of circRNA_09505 on inflammation is investigated in vitro and in vivo by use of macrophage cell models and collagen-induced arthritis (CIA) mice. Luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) are used to confirm the circRNA_09505/miR-6089 ceRNA network predicted by bioinformatics analysis. Compared with controls, the expression of circRNA_09505 is upregulated in peripheral blood mononuclear cells (PBMCs) from patients with RA. The proliferation and cell cycle are significantly promoted when circRNA_09505 is upregulated in macrophages, whereas knockdown of circRNA_09505 inhibits macrophage proliferation and cell- cycle progression. Besides, circRNA_09505 can act as a miRNA sponge for miR-6089 in macrophages, and promote the production of TNF-α, IL-6, and IL-12 through ceRNA mechanism. Moreover, AKT1 is a direct target of miR-6089. CircRNA_09505 can promote AKT1 expression by acting as a miR-6089 sponge via IκBα/NF-κB signaling pathway in macrophages. Most interestingly, knockdown of circRNA_09505 significantly alleviates arthritis and inflammation in vivo in CIA mice. These data support the hypothesis that circRNA_09505 can function as a miR-6089 sponge and regulate inflammation via miR-6089/AKT1/NF-κB axis in CIA mice.

Glioblastoma multiforme (GBM) is the most lethal brain malignancy which involves multi-gene abnormality. Unfortunately, effective therapy against GBM remains lacking. Previously, we found that NRP-1 and its downstream NRP-1/GIPC1 pathway played an important role in GBM. In our study, we further investigated the upstream signaling of NRP-1 to understand how it is regulated. First, we identified that hsa-miR-124-3p was miRNA differentially expressed in GBM and in normal brain tissues by high-throughput sequencing. Then, by dual luciferase reporter gene, we found miR-124-3p can specially bind to the 3'UTR region of the NRP-1 thus suppresses its expression. Moreover, miR-124-3p overexpression significantly inhibited GBM cell proliferation, migration and tumor angiogenesis which resulted in GBM apoptosis and cell cycle arrest, putatively via NRP-1 mediated PI3K/Akt/NFκB pathways activation in GBM cells. Meanwhile, miR-124-3p overexpression also suppressed tumor growth and reduced tumor angiogenesis when targeted by NRP-1 in a PDX model. Furthermore, NRP-1 mAb exerted synergistic inhibitory effects with miR-124-3p overexpression in GBM. Thus, we discovered that miR-124-3p acts as the upstream suppressor of NRP-1 which promotes GBM cell development and growth by PI3K/Akt/NFκB pathway. The miR-124-3p/NRP-1/GIPC1 pathway as a new pathway has a vital role in GBM, and it could be considered as the potential target for malignant gliomas in future.

Human herpesvirus 6 (HHV-6) is an important immunosuppressive and immunomodulatory virus that primarily infects immune cells and strongly suppresses the proliferation of infected cells. However, the mechanisms responsible for the regulation and suppression mediated by HHV-6 are still unknown. In this study, we examined the ability of HHV-6A to manipulate cell cycle progression in infected cells and explored the potential molecular mechanisms. We demonstrated that infection with HHV-6A imposed a growth-inhibitory effect on HSB-2 cells by inducing cell cycle arrest at the G 2 /M phase. We then showed that the activity of the Cdc2-cyclin B1 complex was significantly decreased in HHV-6A-infected HSB-2 cells. Furthermore, we found that inactivation of Cdc2-cyclin B1 in HHV-6A-infected cells occurred through the inhibitory Tyr15 phosphorylation resulting from elevated Wee1 expression and inactivated Cdc25C. The reduction of Cdc2-cyclin B1 activity in HHV-6-infected cells was also partly due to the increased expression of the cell cycleregulatory molecule p21 in a p53-dependent manner. In addition, HHV-6A infection activated the DNA damage checkpoint kinases Chk2 and Chk1. Our data suggest that HHV-6A infection induces G 2 /M arrest in infected T cells via various molecular regulatory mechanisms. These results further demonstrate the potential mechanisms involved in immune suppression and modulation mediated by HHV-6 infection, and they provide new insights relevant to the development of novel vaccines and immunotherapeutic approaches.

Enterovirus 71(EV71) causes recurring outbreaks of hand, foot and mouth disease and encephalitis leading to complications or death in young children. More effective antiviral drugs are needed to prevent or reduce EV71-related disease and complications. However, there are no standard models currently in use to evaluate activity against EV71 infection both in vitro and in vivo. In this study, the activity of ribavirin and pleconaril against EV71 infection was evaluated in two models. An in vitro EV71 infection model was developed in RD cells, and an in vivo EV71 infection model was applied. Ribavirin and pleconaril effectively increased the viability of infected cells. Pleconaril reduced the morbidity and mortality of one-day-old infected mice, but ribavirin did not protect the infected mice. In all, the results demonstrated that infected cells and infected mice can be used to evaluate antiviral activity of ribavirin and pleconaril against EV71 infection in vitro and in vivo.

The etiology of glioma remains unclear so far. Human herpesvirus 6 (HHV-6) might be associated with glioma, but there is no direct evidence to support this. High percentages of HHV-6 DNA and protein were detected in tissue from gliomas, compared with normal brain tissue. In addition, a strain of HHV-6A was isolated from the fluid specimens from glioma cysts. High levels of interleukin 6 (IL-6), interleukin 8 (IL-8), tumor necrosis factor α, and transforming growth factor β (TGF-β) were detected in the cyst fluid specimens from HHV-6-positive patients with glioma. Furthermore, HHV-6A infection promoted IL-6, IL-8, and TGF-β production in astrocyte cultures. Our studies strongly suggest the involvement of HHV-6 infection in the pathogenesis of glioma.