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The 2-oxoglutarate (2OG)-dependent non-heme enzyme FtmOx1 catalyzes the endoperoxide biosynthesis of verruculogen. Although several mechanistic studies have been carried out, the catalytic mechanism of FtmOx1 is not well determined owing to the lack of a reliable complex structure of FtmOx1 with fumitremorgin B. Herein we provide the X-ray crystal structure of the ternary complex FtmOx1⋅2OG⋅fumitremorgin B at a resolution of 1.22 Å. Our structures show that the binding of fumitremorgin B induces significant compression of the active pocket and that Y68 is in close proximity to C26 of the substrate. Further MD simulation and QM/MM calculations support a CarC-like mechanism, in which Y68 acts as the H atom donor for quenching the C26-centered substrate radical. Our results are consistent with all available experimental data and highlight the importance of accurate complex structures in the mechanistic study of enzymatic catalysis., (© 2021 Wiley-VCH GmbH.)

Regression models with a functional response and functional covariate have received significant attention recently. While various nonparametric and semiparametric models have been developed, there is an urgent need for model selection and diagnostic methods. In this article, we develop a unified framework for estimation and model selection in nonparametric function-on-function regression. We propose a general nonparametric functional regression model with the model space constructed through smoothing spline analysis of variance (SS ANOVA). The proposed model reduces to some of the existing models when selected components in the SS ANOVA decomposition are eliminated. We propose new estimation procedures under either L 1 or L 2 penalty and show that the combination of the SS ANOVA decomposition and L 1 penalty provides powerful tools for model selection and diagnostics. We establish consistency and convergence rates for estimates of the regression function and each component in its decomposition under both the L 1 and L 2 penalties. Simulation studies and real examples show that the proposed methods perform well. Technical details and additional simulation results are available in online supplementary materials.

Porcine circovirus type 2 (PCV2) causes increased mortality and poor growth or weight loss in apparently healthy swine. Therefore, methods to detect PCV2-specific antibodies in swine serum are important for prevention, diagnosis, and control of PCV2-associated diseases (PCVAD). In this study, PCV2 virus-like particles (VLPs) were used to develop a rapid, simple and economical indirect enzyme-linked immunosorbent assay to detect (with high sensitivity) PCV2-specific antibodies in swine serum. The PCV2 capsid protein (Cap) was overexpressed in E. coli after optimizing the cap gene. Subsequently, the soluble Cap was rapidly purified in one step by automated fast protein liquid chromatography (FPLC). The purified PCV2 Cap was shown by transmission electron microscopy and gel filtration chromatography to be capable of self-assembling into VLPs in vitro. Using the purified VLPs as antigens, optimal operating conditions for the VLP ELISA were determined. The concentration of PCV2 VLPs was 1 µg/ml per well, and the dilution factors for swine serum and horseradish peroxidase (HRP)-labeled goat anti-pig antibody were 1:150 and 1:4000, respectively. Out of 241 serum samples tested with this assay, 83.4 % were found to be positive. Importantly, the VLP ELISA had a total coincidence rate of 97.4 % (74/76) compared to an Ingezim PCV2 ELISA IgG assay. In summary, this rapid, inexpensive VLP ELISA has the potential to greatly facilitate large-scale investigations of PCV2-associated serotypes.

Objective: We studied the antiviral activity of probiotics and possible mechanism thereof., Methods: With hemagglutination in vitro and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) of chicken embryo fibroblast (CEF), the hemagglutination titers and virus inhibition rate were respectively determined to evaluate the impact of probiotics on Newcastle Disease Virus (NDV)., Results: Five probiotics and their metabolites could reduce the viral titer of NDV, but the two pathogenic bacteria and their metabolites could not. These results indicated that probiotics may destruct the structure of NDV directly and this effect was strain-specific. Probiotics could inhibit the replication rate of NDV significantly in a dose-dependent manner (P<0.01). The inhibitory rate increased when the cells were pre-treated with probiotics, which indicated that the probiotics might block NDV adsorbing to the cells. The fact that addition of probiotics and NDV at the same time reduced the cytopathic effect (CPE) of cells suggested that probiotics could destruct the viral structure directly; the lower inhibitory rate of viral replication when cells infected with NDV before addition of probiotics to the cells suggested that the probiotics worked hardly after the cells were infected., Conclusion: Probiotics can not only destruct the virus, but also block the virus infection of the cells and inhibit the viral proliferation in the cells.