Establishment, validation and application of fluorescence focus assay for rapid titration of Japanese encephalitis virus / 中华微生物学和免疫学杂志

Objective:To establish and validate a fluorescence focus assay (FFA) for rapid titration of Japanese encephalitis virus (JEV) and to evaluate its application in the production of Japanese encephalitis vaccine.Methods:Recombinant JEV non-structural protein 1 (NS1) was expressed in a prokaryotic expression system. After purification, JEV-NS1 was used to immunize rabbits to induce polyclonal antibody. FFA was established with the polyclonal antibody to titer JEV. The accuracy of FFA was validated by comparing with plaque assay, and the specificity, precision, linearity, range and robustness of FFA were also validated. Twenty-eight batches of live-attenuated Japanese encephalitis vaccine were titrated with FFA and plaque assay to analyze the relationship between the two assays.Results:FFA established with polyclonal antibody against JEV-NS1 could be used to titrate JEV, and there was no cross reaction with other viruses (tick-borne encephalitis virus, yellow fever virus, coxsackievirus A2, coxsackievirus A4). Results of the validation tests showed that FFA met the requirement of quality control for live-attenuated Japanese encephalitis vaccine. FFA was more consistency than plaque assay.Conclusions:The established FFA could be used for virus titration in the production of live-attenuated Japanese encephalitis vaccine.

SPA0355 prevents ovariectomy-induced bone loss in mice

Estrogen withdrawal in post-menopausal women leads to overactivation of osteoclasts, which contributes to the development of osteoporosis. Inflammatory cytokines are known as one of mechanisms of osteoclast activation after estrogen deficiency. SPA0355 is a thiourea derivative that has been investigated for its antioxidant and anti-inflammatory activities. However, its efficacy in bone resorption has not been previously investigated. The aim of this study was to investigate the impact of SPA0355 on the development of osteoporosis and to explore its mode of action. In vitro experiments showed that SPA0355 inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis in primary bone marrow-derived macrophages. This effect appears to be independent of estrogen receptor activation as ICI 180,782 failed to abrogate its effects on osteoclasts. Further signaling studies revealed that SPA0355 suppressed activation of the MAPKs, Akt, and NF-κB pathways. SPA0355 also increased osteoblastic differentiation, as evidenced by its effects on alkaline phosphatase activity and mineralization nodule formation. Intraperitoneal administration of SPA0355 to ovariectomized mice prevented bone loss, as verified by three-dimensional images and bone morphometric parameters derived from µCT analysis. Noticeably, SPA0355 did not show hepatotoxicity and nephrotoxicity and also had little effect on hematological parameters. Taken together, the results indicate that SPA0355 may protect against bone loss in ovariectomized mice by stimulation of osteoblast differentiation and by inhibition of osteoclast resorption. Therefore, SPA0355 is a safe and potential candidate for management of postmenopausal osteoporosis.

Feeding characteristics of an amoeba (Lobosea: Naegleria) grazing upon cyanobacteria: food selection, ingestion and digestion progress.

Bacterivory by heterotrophic nanoflagellates and ciliates has been widely studied in aquatic environments, but data on the grazing of amoebae, are still scarce. From the water samples of Dianchi Lake (Kunming, Yunnan Province, China), we isolated an amoeba, designated as Naegleria sp. strain W2, which had potent grazing effects on some kind of cyanobacteria. The food selection mechanism and the digestion process of the amoeba were investigated in batch experiments. Predation experiments showed that filamentous cyanobacteria (e.g., Anabaena, Cylindrospermum, Gloeotrichia, and Phormidium) were readily consumed, with clearance rates ranging from 0.332 to 0.513 nL amoeba(-1) h(-1). The tight threads (Oscilltoria) and aggregates (Aphanizomenon) could not be ingested; however, their sonicated fragments were observed inside food vacuoles, suggesting that their morphologies prevent them from being ingested. Live video microscopy noted that unicellular Chroococcaceae (e.g., Synechococcus, Aphanocapsa, and Microcystis) were excreted after ingestion, indicating that food selection takes place inside food vacuoles. To determine whether the tastes or the toxins prevented them from being digested, heat-killed cells were retested for predation. Digestion rates and ingestion rates of the amoebae for filamentous cyanobacteria were estimated from food vacuole content volume. Through a "cold-chase" method, we found that the food vacuole contents declined exponentially in diluted amoebae cells, and digestion rates were relatively constant, averaging about 1.5% food vacuole content min(-1) at 28 degrees Celsius. Ingestion strongly depended on the satiation status of the amoebae, starved amoebae fed at higher rates compared with satiated amoebae. Our results suggest that the food selection and food processing mechanisms of the amoeba are similar to those of interception feeding flagellates; however, filamentous cyanobacteria cannot obtain a refuge under the grazing pressure of phagotrophic amoebae, which may widen our knowledge on the grazing of protists.