Search

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate–NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc‐based secretion assay features a very high signal‐to‐noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384‐well microplates. We further developed a split NanoLuc‐based assay that enables the real‐time monitoring of type III secretion‐dependent injection of effector–HiBiT fusions into host cells stably expressing the complementing NanoLuc–LgBiT. [ABSTRACT FROM AUTHOR]

In the present study, average 48.19±1.12% of fillets (trimmed and without skin) was obtained from the round fish (Lates calcarifer) and rest 51.80±1.13% remained as filleting waste. Protein soluble (3% protein concentration + 1.5% brine) prepared from filleting waste was used at 4 different levels of incorporation like 0% (C), 5% (TI1), 10% (TI2) and 15% (TI3) of the weight of fillets using an injector. Weight gain, drip loss, fillet yield, cooking yield of chilled fillets were investigated during 12 days chilled storage. Significantly highest (p<0.05) weight gain of fillets was observed in TI2 sample whereas TI1 and TI3 showed comparatively lower weight gain. Drip loss of all the injected fillets increased dramatically with storage time in chilling conditions and drip loss was significantly different at different levels of protein injection. Fillets injected with no protein (C) demonstrated significantly (p<0.05) highest drip loss during the storage time and lowest in TI2. Fillet yield (%) during chilled storage was found to have significantly decreased in all treatments including control with highest fillet yield in TI2 sample. Cooking yield (%) of fillets during chilled storage was found to have significantly decreased (p<0.05) for all treatments including control. At the end of 12 days chilled storage, samples TI1, TI2 and TI3 showed significantly higher (p<0.05) than control. Based on these results, injection of protein soluble into Seabass fillets is considered as an effective means to improve the yield of fillets. [ABSTRACT FROM AUTHOR]