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Cell envelope proteases (CEPs) can break down milk protein into peptides with different functions, which are of great benefit to human health. Therefore, the high-yield CEPs of Lactobacillus plantarum have the potential to produce functional dairy products. In previous experiments, we found that Na2HPO4, inulin, casein peptone and leucine have significant effects on CEP production by Lactobacillus plantarum LP69. So we proceeded to optimize the composition of the CEP-producing culture medium of L. plantarum through Box-Behnken design and response surface methodology. The protease activity, protein content and specific activity of CEPs produced by L. plantarum by inulin (0.2, 0.3, 0.4 %), casein peptone (0.4, 0.6, 0.8 %), Na2HPO4 (0.50, 0.52, 0.54 %) and leucine (14, 16, 18 mg/L) were evaluated. The optimal ratio of medium is 0.4 % inulin, 0.66 % casein peptone, 0.5 % Na2HPO4 and 14.04 mg/L Leucine. The final enzyme activity reached (24.46±0.81) U/mL, and the specific activity reached (1.41±0.46) U/mg.

Menstrual blood-derived endometrial stem cells (MenSCs) have attracted increasing interest due to their excellent safety, and lack of ethical dilemma as well as their ability to be periodically obtained in a noninvasive manner. However, although preclinical research as shown the therapeutic potential of MenSCs in several diseases, their poor cell survival and low engraftment at disease sites reduce their clinical efficacy. Flotillins (including Flot1 and Flot2) are implicated in various cellular processes, such as vesicular trafficking, signal transduction, cell proliferation, migration and apoptosis. In this study, we aimed to determine the effects of Flotillins on MenSCs survival, proliferation and migration. Our experimental results show that MenSCs were modified to overexpress Flot1 and/or Flot2 without altering their intrinsic characteristics. Flot1 and Flot2 co-overexpression promoted MenSC viability and proliferation capacity. Moreover, Flot1 or Flot2 overexpression significantly promoted the migration and inhibited the apoptosis of MenSCs compared with the negative control group, and these effects were stronger in the Flot1 and Flot2 gene co-overexpression group. However, these effects were significantly reversed after Flot1 and/or Flot2 knockdown. In conclusion, our results indicate that Flot1 and Flot2 overexpression in MenSCs improved their proliferation and migration and inhibited their apoptosis, and this might be an effective approach to improve the efficiency of cell-based therapies. [ABSTRACT FROM AUTHOR]

In present study, the effects of carbon sources (glucose, lactose, sucrose, galactose, maltose and soluble starch) and nitrogen sources (casein peptone, whey protein, soy peptone, yeast, tryptone, beef extract and peptone) on activity of cell envelope proteinases (CEP), specific activity, protein content, OD600 value and pH in MRS broth fermented by Lactobacillus plantarum LP69 were investigated by individual factor experiment. The results indicated that carbon and nitrogen sources have significant influence on the activity of CEP and specific activitys of L. plantarum LP69, glucose, maltose, casein peptone and peptone are superior to other selected carbon and nitrogen sources. The optimum concentrations of glucose, maltose, casein peptone and peptone for L. plantarum LP69 are 2%, 2%, 1% and 1%; the activity of CEP are 19.52U/mL, 21.13U/mL, 13.49U/mL and 20.61U/mL, respectively.

Niemann-Pick disease type C1 (NPC1) is a hereditary neurodegenerative disorder caused by a mutation in the NPC1 gene. This gene encodes a transmembrane protein found in lysosomes. This disease characterized by hepatosplenomegaly, neurological impairments and premature death. Recent preclinical studies have shown promising results in using mesenchymal stem cells (MSCs) to alleviate the symptoms of NPC1. One type of MSCs, known as human menstrual blood-derived endometrial stem cells (MenSCs), has attracted attention due to its accessibility, abundant supply, and strong proliferation and regeneration capabilities. However, it remains uncertain whether the conditioned medium of MenSCs (MenSCs-CM) can effectively relieve the symptoms of NPC1. To investigate this further, we employed the CRISPR-Cas9 technique to successfully create a Npc1 gene knockout N2a cell line (Npc1KO N2a). Sanger sequencing confirmed the occurrence of Npc1 gene mutation in these cells, while western blotting revealed a lack of NPC1 protein expression. Filipin staining provided visual evidence of unesterified cholesterol accumulation in Npc1KO N2a cells. Moreover, Npc1KO N2a cells exhibited significantly decreased viability, increased inflammation, and heightened cell apoptosis. Notably, our study demonstrated that the viability of Npc1KO N2a cells was most significantly improved after being cultured by 36 h-collected MenSCs-CM for 0.5 days. Additionally, MenSCs-CM exhibited the ability to effectively reduce inflammation, counteract cell apoptosis, and ameliorate unesterified cholesterol accumulation in Npc1KO N2a cells. This groundbreaking finding establishes, for the first time, the protective effect of MenSCs-CM on N2a cells with Npc1 gene deletion. These findings suggest that the potential of MenSCs-CM as a beneficial therapeutic approach for NPC1 and other neurodegenerative diseases. [ABSTRACT FROM AUTHOR]