Isolation and Purification of Bacterial Extracellular Vesicles from Human Feces Using Density Gradient Centrifugation.

Bacterial extracellular vesicles (BEVs) are nanovesicles derived from bacteria that play an active role in bacteria-bacteria and bacteria-host communication, transferring bioactive molecules such as proteins, lipids, and nucleic acids inherited from the parent bacteria. BEVs derived from the gut microbiota have effects within the gastrointestinal tract and can reach distant organs, resulting in significant implications for physiology and pathology. Theoretical investigations that explore the types, quantities, and roles of BEVs derived from human feces are crucial for understanding the secretion and function of BEVs from the gut microbiota. These investigations also necessitate an improvement in the current strategy for isolating and purifying BEVs. This study optimized the isolation and purification process of BEVs by establishing two density gradient centrifugation (DGC) modes: Top-down and Bottom-up. The enriched distribution of BEVs was determined in fractions 6 to 8 (F6-F8). The effectiveness of the approach was evaluated based on particle morphology, size, concentration, and protein content. The particle and protein recovery rates were calculated, and the presence of specific markers was analyzed to compare the recovery and purity of the two DGC modes. The results indicated that the Top-down centrifugation mode had lower contamination levels and achieved a recovery rate and purity similar to that of the Bottom-up mode. A centrifugation time of 7 h was sufficient to achieve a fecal BEV concentration of 108/mg. Apart from feces, this method could be applied to other body fluid types with proper modification according to the differences in components and viscosity. In conclusion, this detailed and reliable protocol would facilitate the standardized isolation and purification of BEVs and thus, lay a foundation for subsequent multi-omics analysis and functional experiments.

Exploring design features for enhancing players' challenge in strategy games.

This paper examines how to make a player feel more challenged in a strategic computer game. It is hypothesized that information availability and resource advantage affect play difficulty, which in turn affects the challenge experienced. The difficulty of play can be defined in terms of the mental workload that players experience and the physical effort that players exert. Forty-five male college and graduate students participated in a 3 x 3 (information availability x resource advantage) between-subjects factorial design experiment. This experiment measured player mental workload, physical effort, and challenge. The results indicate that information availability affects player mental workload, and resource advantage affects levels of player physical effort, respectively. Moreover, the relationship between mental workload and challenge was found to be an inverted U-shaped curve; in other words, too much or too little mental workload may decrease player challenge. The relationship between physical effort and challenge exhibited similar characteristics.