Grail is a crucial regulator of various biological processes, including the development of T-cell anergy, antiviral innate immune response, and cancer. However, the role of Grail in adipogenesis and obesity remains unclear. Here, we demonstrated that Grail knockdown in vitro leads to a decrease in PPARγ expression, resulting in adipogenesis inhibition. However, Grail overexpression induced the same effects. Grail was shown to interact with PPARγ, targeting it for degradation and modulating its adipogenic activity. PPARγ expression was shown to be considerably reduced in Grail knockout (KO) mice fed normal diet or high-fat diet (HFD). The administration of both normal diet or HFD to Grail KO mice led to lower adipose mass and body weight than those in the wild-type mice. HFD-fed Grail KO mice had improved glucose and insulin tolerance. Taken together, our results indicate that Grail plays a pivotal role in adipogenesis and diet-induced obesity by regulating PPARγ activity.

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The outbreak of severe acute respiratory syndrome (SARS) was caused by a newly identified coronavirus (SARS-CoV) in 2003. To detect early SARS-CoV infection, a one-step, real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay was developed that could simultaneously detect nucleocapsid (N), membrane (M), and spike (S) genes of SARS-CoV with the same PCR condition using either Applied Biosystems (ABI) Prism 7700 Sequence Detection System or Roche LightCycler. The sensitivity of this assay was evaluated using cell culture-derived viruses, in vitro transcribed viral RNA, and clinical specimens. The SARS-S, -M, and -N primer/probe sets described in this paper could detect one to ten copies of in vitro transcribed S, M, and N RNA per test using both the ABI and Roche assay systems. The relative sensitivities for detecting cell culture-derived SARS-CoV were 0.01, 0.01, and 0.001 PFU/test, respectively. It showed that SARS-N has comparable detection efficiencies to SARS2 and SARS3 which are primers sets designed by Centers for Disease Control and Prevention. In addition, SARS-S and SARS-M also demonstrated equivalent sensitivity to the commercially available RealArt HPA-Coronavirus reagents (Artus). The relative sensitivity of these primer/probe sets was also examined using human sera spiked viruses and clinical specimens from four confirmed SARS patients. Similar results as above were obtained. Specificity tests and sequence alignment showed that these primer/probe sets annealed perfectly to 31 isolates of SARS-CoV; and there was no cross detection with other coronaviruses and human respiratory tract-associated viruses. Therefore, not only is it compatible with the ABI and Roche systems, this multiple-gene detection assay also has the merit of being a rapid, safe, sensitive, and specific tool for accurate diagnosis of SARS-CoV infection.

The spread of chikungunya virus (CHIKV) is reaching pandemic levels, and vaccines and antivirals to control CHIKV infection have yet to be approved. Virus-like particles (VLPs), a self-assembled native multi-subunit protein structure, could potentially be used as an antigen for serological detection and vaccine development. In the current study, we describe the production of novel CHIKV VLPs from mosquitoes using a Baculovirus/Mosquito (BacMos) system in a simple Biosafety Level‐2 laboratory. Substantial envelope and capsid protein secretions were detected in culture medium. Co-fractionation of CHIKV E2, E1, and capsid proteins via sucrose gradient ultracentrifugation provided evidence of VLP formation. Transmission electron microscopy and dynamic light scattering analysis revealed the formation of VLPs in the form of spherical particles with a diameter of roughly 40 nm in transduced cells and culture medium. VLP-based IgM capture ELISA in CHIKV patient sera revealed native epitopes on the VLPs. These non-purified VLPs were shown to act as an antigen in CHIKV-specific IgM capture ELISA. The immunization of CHIKV-VLPs alone in mice induced a balance CHIKV-specific IgG2a/IgG1 antibodies and neutralized antibody responses. The study provides support for the hypothesis that mosquito cell-derived CHIKV VLPs could serve as a novel antigen for serological detection and the development of vaccines against CHIKV infection. Key points • CHIKV VLPs secreted from BacMos-CHIKV 26S-transduced mosquito cell. • This CHIKV VLPs potentially serve as an alternative capture antigen for MAC-ELISA. • Unadjuvanted CHIK VLPs induce CHIKV-specific IgG and NT responses in mice.

Multiple organ dysfunction caused by hyperinflammation remains the major cause of mortality during sepsis. Excessive M1-macrophage activation leads to systemic inflammatory responses. Gene related to anergy in lymphocytes (Grail) is regarded as an important regulator of T cells that functions by diminishing cytokine production. However, its role in regulating macrophage activation and organ injury during sepsis remains unclear. Our aim was to examine the effects of Grail on macrophage reactivity and organ injury in endotoxemic animals. Wild-type and Grail knockout mice were injected with vehicle or Escherichia coli lipopolysaccharide and observed for 24 h. Changes in blood pressure, heart rate, blood glucose, and biochemical variables were then examined. Moreover, levels of neutrophil infiltration, MMP-9, and caspase 3 were analyzed in the lungs of animals. The expression of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages stimulated with LPS were also assessed in the presence or absence of Grail. Results indicated that loss of Grail expression enhances the induction of pro-inflammatory cytokines in J774A, RAW264.7, and primary peritoneal macrophages treated with LPS. Furthermore, LPS-induced macrophage hyperactivation was alleviated by ectopic Grail overexpression. In vivo studies showed that Grail deficiency exacerbates organ damage in endotoxemic animals. Levels of neutrophil infiltration, MMP-9, and caspase 3 were significantly increased in the lungs of Grail-deficient endotoxemic mice. Thus, these results suggest that Grail contributes to the attenuation of hyperinflammation caused by activated macrophages and prevents organ damage in endotoxemic mice. We suggest that Grail signaling could be a therapeutic target for endotoxemia.