Construction of a food-grade gene editing system based on CRISPR-Cas9 and its application in Lactococcus lactis NZ9000.

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system has been widely used in gene editing of various organisms. However, food-grade gene editing systems in lactic acid bacteria are still preliminary. Red/ET-dependent homologous recombination or CRISPR-based systems have been developed to gene editing in Lactococcus lactis, but these methods are overall inefficient. In the present study, a recombinant system based on CRISPR/Cas9 technology combined with Red/ET was developed using the plasmid pMG36e derived from Lactococcus lactis. Then, the developed recombinant system was applied to Lactococcus lactis. Knockout efficiency was significantly higher using the developed system (91%). In addition, this system showed the potential to be used as a high-throughput method for hierarchical screening. Finally, a gene-edited strain was obtained, and no antibiotics or exogenous genes were introduced using the developed gene editing system. Thus, the efficient system in lactic acid bacteria was constructed and optimized.

Food-grade expression of nattokinase in Lactobacillus delbrueckii subsp. bulgaricus and its thrombolytic activity in vitro.

OBJECTIVES: To produce nattokinase in a food-grade expression system and evaluate its thrombolytic activity in vitro. RESULTS: No nattokinase activity from reconstituted strains was observed in simulated gastric juice, but the enzyme was stable in intestinal fluid, the relative activity of which was found to be 60% after 4 h. Due to the nattokinase being produced intracellularly by recombinant bacterial strains, the persistence of the bacteria in gastric juice ensured transmission of the nattokinase into intestinal juice. Because of subsequent disintegration of the bacteria, the highest nattokinase activity was observed after 3 h at approximately 32%, following its carriage within the recombinant strains to the intestinal fluid. CONCLUSIONS: This study demonstrated that nattokinase from recombinant strains exhibited good thrombolytic activity in vitro and may be used by the dairy fermentation industry for the development of novel thrombolytic functional foods.

Factors, components and dynamics: investigation of journal self-citation and citation by equal opportunity model.

The effectivity of journal impact factor (JIF) is questioned in evaluating academic players. Coercive self-citation was widely criticized and Clarivate annually suppressed journals with high journal self-citation (JSC) rates. Recently, some journals significantly increased their articles but their JIFs and JSC rates were diversified which lacks reasonable explanations. Here, we revealed the complexities of the dynamical interactions among different influence factors and different components of journal citation. Journal citation frequencies have strong correlations with JSC rates regarding citable items, significant correlations with numbers of journals' articles and negative significant correlations with JSC rates regarding total citations. Journal citation consists of JSC, intradisciplinary non-JSC and interdisciplinary non-JSC. JSC has the quickest dynamic and interdisciplinary non-JSC has the slowest dynamic while most journals are more cited by interdisciplinary citations. Journal citation is initially decided by the number of citable items. Journal's share (citing articles) in related disciplines influence JSC and intradisciplinary non-JSC positively and negatively, respectively. Multiple citations promoted by the increase in articles, the skewness of the topic profile, preference-related self-citation and anomalous self-citation are of benefit to citation. The complex dynamical interactions result in increment effects of the number of articles acting on JIF, which is stronger for JSC and citable items. The increase in articles also promotes intradisciplinary non-JSC when the journal's shares in related disciplines are low. This process will be reversed after the journal's shares become high enough which may finally decrease its impact factor. It is quicker for the journal with a stronger intradisciplinary citation. The calculational results of the average JIFs of selected journals agree with the statistical results. In addition, we can explain the related situations of some journals. Finally, we suggested that the JSC rate in terms of citable items should be considered for judging the level of JSC.

Study of the Oxidation Behavior of Fine-Grained Graphite ET-10 by Combining X-ray µCT with Mercury Porosimetry.

By combining X-ray micro-computed tomography with mercury porosimetry, the evolution of the oxygen supply, porous structure, mass loss and oxidized compositions were investigated to characterize the oxidation behavior of fine-grained graphite ET-10, regarding the geometry of the specimen and its oxidation temperature. Here, the porous structure and the gas flows out of and into the porous structure were comprehensively compared for two kinds of specimens-large pure graphite (D = H = 25.4 mm), oxidized at a test facility based on ASTM D7542, and small partially SiC-coated graphite (D ≈ 1 mm and H = 1.95 mm), oxidized in the bottom section of a U-type tube. The fine grains and large geometry resulted in small pores and long flow distances, which exhausted the oxygen in the small stream to the interior of the specimen, making its oxidation deviate from the kinetics-controlled regime. In addition, the well-known three-regime theory was reasonably reinterpreted regarding the oxidation of different compositions, binders and fillers. The kinetics-controlled uniform oxidation mainly oxidizing binders is restricted by their limited contents, while the rate of surface-dominated oxidation increases continuously via the consumption of more fillers. Furthermore, we proposed a new design for the test facility used for the oxidation experiment, wherein a partially shielded millimeter specimen can be oxidized in the long straight bottom section of a U-tube, and this will be discussed further in related future studies.

Characterizing thermal-oxidation behaviors of nuclear graphite by combining O2 supply and micro surface area of graphite.

The effects of different parameters on oxidation rate are non-linear, interactive and diversified in which the change of adequacy of O2 supply is an important indicator. The influence of microstructure on oxidation rate became stronger worsening the fitting linearity to calculate the activation energy based on present method with the decreased adequacy of O2 supply due to the increase of temperature, the decrease of gas flow rate, etc. Here, we proposed a method to characterize thermal-oxidation behaviors of nuclear graphite by combining O2 supply and micro surface area of graphite. The proposed method improved the linearity and reduced the standard error of Arrhenius plots of oxidized graphite IG-110 (10 L/min reactant gas) and ET-10 (0.2 L/min reactant gas). The value of activation energy of graphite IG-110 oxidized under ASTM D7542 condition is calculated as 220 kJ/mol by this method echoing the results of previous studies with sufficient O2 supply. For the conditions with less O2 supply at low gas flow rate and/or high temperature, the change of microstructure of oxidized graphite should be obtained as an important factor influencing oxidation rate of graphite.