Rapid and Highly Sensitive Method for Evaluating Surface Coatings against an Enveloped RNA Virus.

The COVID-19 pandemic has increased public health vigilance worldwide. The coronavirus (SARS-CoV-2) can spread via aerosols, and droplet-borne viruses remain viable on nonliving surfaces for long duration. Hence, effective antiviral coatings are highly useful in eliminating viral persistence on nonliving surfaces. Although innovative antiviral coatings have been designed, conventional procedures for antiviral assays are generally laborious, time-consuming, and have a high limit of detection. In the present study, we report a rapid and highly sensitive method for evaluating antiviral coatings by measuring the luciferase activity derived from recombinant Sendai virus (SeV). The physicochemical characteristics of SeV, which has a single-stranded RNA genome encapsulated within a lipid envelope, allow us to exploit it as an indicator of the physicochemical potential of coating materials against enveloped RNA viruses in general. We demonstrate that SeV-based assay systems allow for the rapid and quantitative evaluation of the surface coatings composed of iodine solubilized in polyvinyl acetate. Additionally, we have investigated the effect of mucins, the dominant protein component of saliva, on the antiviral activity of surface coatings. The presence of mucins in the SeV suspension considerably rescues luciferase activity at the viral-surface interface, presumably due to mucin-mediated viral protection. Our findings provide insights into a procedure capable of the rapid evaluation and optimization of surface coatings, and suggest an important role of the mucin in the valid evaluation of antiviral agents.

Acetoacetate is a trigger of NLRP3 inflammasome activation in bovine peripheral blood mononuclear cells.

Repeat breeding, which is non-pregnancy following three or more breeding attempts, is a serious reproductive disorder in cattle. In the present study, metabolomic profiling was used to identify metabolites in the blood plasma of repeat breeder cows (RBCs) and non-RBCs. Metabolomic analysis showed that acetoacetate (AcAc), a ketone body, was detected in RBCs, but not in non-RBCs. In contrast, ß-hydroxybutyrate (BHB) was at similar levels in both RBCs and non-RBCs. We hypothesized that an imbalance of AcAc and BHB induces abnormal inflammatory conditions, especially the NLRP3 inflammasome, which regulates sterile inflammation to control interleukin (IL)-1ß secretion, and may be associated with repeat breeding in cattle. To investigate this hypothesis, blood samples were collected from both non-RBCs and RBCs on day 7 of the estrous cycle. The mRNA expression of IL1B in peripheral blood mononuclear cells (PBMCs) was observed to be higher in RBCs than in non-RBCs. To test the effects of AcAc and BHB on inflammatory responses, blood samples were collected from healthy cows and PBMCs were isolated. PBMCs were treated with AcAc and BHB to investigate the activation of the NLRP3 inflammasome (complex of NLRP3, ASC, and caspase-1) and IL-1ß secretion. AcAc treatment resulted in higher protein and/or mRNA expression of NLRP3 and IL-1ß in PBMCs. Moreover, AcAc increased the co-localization of NLRP3 and ASC and stimulated caspase-1 activation, indicating the formation of the platform of the NLRP3 inflammasome. Addition of specific NLRP3 inhibitor, MCC950, suppressed AcAc stimulation-induced IL-1ß secretion. Contrary to the effects of AcAc, BHB treatment suppressed the activation of NLRP3 inflammasome and IL-1ß secretion in response to AcAc and typical NLRP3 inflammasome triggers. These findings demonstrate that AcAc can potentially trigger NLRP3 inflammasome activation, resulting in IL-1ß secretion, and that these inflammatory responses are suppressed by BHB in bovine PBMCs. In addition, the imbalance between AcAc and BHB with higher levels of IL-1ß may be associated with repeat breeding in cattle.

The transfer of parthenogenetic embryos following artificial insemination in cows can enhance pregnancy recognition via the secretion of interferon tau.

Repeat breeding is a reproductive disorder in cattle. Embryo transfer following artificial insemination (AI) improves pregnancy rate by replenishing interferon tau (IFNT), but it results in a notably higher rate of twin occurrence. This study hypothesized that parthenogenetic (PA) embryo transfer following AI (AI + PA) could improve the conception rate because that PA embryo become as a supplemental source of IFNT without twins. PA embryos showed higher IFNT mRNA expression than in vitro fertilization (IVF) embryos. An examination of the effect of the cultured conditioned media (CM) of PA or IVF embryos on Madin-Darby bovine kidney cells with stably introduced promoter-reporter constructs of interferon-stimulated gene 15 (ISG15, marker of IFN response) showed higher stimulation levels of ISG15 promoter activity with PA than with IVF embryo. We investigated in vivo the effect of AI + PA on healthy Japanese Black cattle. Cattle transferred with PA embryo alone were non-fertile, but those that underwent AI + PA showed a pregnancy rate of 53.3%, the similar as that with AI alone (60%). In pregnant cattle in AI + PA group, adding the PA embryo upregulated the expression of ISGs and plasma progesterone concentration. No twin were generated in AI only and AI + PA groups. Using repeat breeding Holstein cows that did not become pregnant with 4-9 times of AI, transfer of PA embryo following AI resulted in a higher pregnancy rate than that of control (AI only). We suggest that AI + PA may be beneficial for improving maternal pregnancy recognition in repeat breeder cattle while avoiding twin generation.