L-Asparaginase (ASNase), an aminohydrolase, is widely utilized in the pharmaceutical and food industries. Among its various sources, Escherichia coli K12-derived EcASNase has been employed as a clinical drug for the treatment of acute lymphoblastic leukemia (ALL). However, the limited catalytic activity and stability of EcASNase have restricted its broader application in medicine and food processing. In this study, a random mutagenesis library was constructed via error-prone PCR, followed by high-throughput screening using a coupled bacterial growth strategy. Three positive mutants with enhanced activity were identified: G38S, Q212Y, and S274P, exhibiting activities 1.4-, 1.1-, and 1.2-fold higher than the wild type (WT), respectively. Saturation mutagenesis libraries were subsequently generated for positions 38, 212, and 274, leading to the identification of mutants G38A, G38S, G38Q and G38V, with k_cat/K_m values 1.7-, 1.5-, 2.1-, and 2.2-fold higher than WT, respectively. Among these, G38V emerged as the most active mutant, with a T_m value increased by 8.4 ℃ compared to WT. Combination mutations, such as G38V/Q212F and G38V/S274P, failed to yield further activity improvements. This research elucidates the contributions of critical residues to the enzyme's activity and stability, providing novel insights into the rational design and development of therapeutic enzymes.

Based on mass spectrometry imaging method, we investigated the effects of Panax notoginseng in improving diabetic retinopathy (DR) and interfering with corneal, vitreous and retinal metabolites, to reveal the mechanism of Panax notoginseng's action in improving DR. All animal experiments were approved by the Experimental Animal Ethics Committee of Beijing University of Chinese Medicine (Approval No.: BUCM-2023052204-2117). Streptozotocin (STZ)-induced diabetes mellitus (DM) rat model was used, and fasting blood glucose (FBG) and glucosylated serum protein (GSP) levels were measured in each group of rats. Occludin and zonula occludens-1 (ZO-1) were detected by immunofluorescence staining; air flow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was used to detect endogenous metabolites in the cornea, vitreous, and retinal microregions of the eyes of rats in the DM group and Panax notoginseng group. Endogenous metabolites were detected in the cornea, vitreous, and retinal microregions of the DM and Panax notoginseng groups, and the DM and Panax notoginseng groups were screened for different metabolites by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). Differential metabolites were screened in the DM and Panax notoginseng groups, the in situ spatial information of differential metabolites in each microregion was analyzed, and the related metabolic pathways were analyzed by the Kyoto encyclopedia of genes and genomes (KEGG) database. The results showed that compared with the DM group, diabetic rats in the Panax notoginseng group showed a decreasing trend in both FBG and GSP, and an increase in the expression of ZO-1 and occludin in the retina (P < 0.001); AFADESI-MSI analysis showed that there were a total of 34 differential metabolites in the cornea, vitreous body, and retinal microregion in the Panax notoginseng group, of which Panax notoginseng called back 13 differential metabolites. In the retinal microregion, Panax notoginseng significantly regulated lysophosphatidylserine (18∶0), phosphatidylethanolamine (34∶2) and phosphatidylserine (40∶7/42∶7). The metabolic pathway enrichment results indicated that Panax notoginseng mainly regulated glycerophospholipid metabolism, glycosylphosphatidylinositol synthesis, niacin and nicotinamide metabolism as well as glycerol ester metabolic pathways. In conclusion, Panax notoginseng improves the blood-retinal barrier (BRB) in diabetic rats, and its mechanism of action may be closely related to glycerophospholipid metabolism. This study provides scientific evidence for the mechanism of action of Panax notoginseng in improving DR, and demonstrates the potential of mass spectrometry imaging technology applied to the study of pharmacological mechanisms.

Nutlin-3 is a representative small molecule MDM2-p53 antagonist, which can stabilize the p53 state by disrupting the interaction between p53 and MDM2, thereby inducing the p53 signaling pathway to exert antitumor effects. In this study, six wild-type p53 tumor cell lines, HCT-116, H460, HepG2, MCF-7, A549 and SJSA-1, were used as research objects, and the effects of nutlin-3 on the proliferation of six wild-type p53 cancer cells were detected by methyl thiazolyl tetrazolium (MTT) method and plate cloning assay. The effects of nutlin-3 on H460 cell cycle and apoptosis were detected by flow cytometry. Western blot assay was used to detect ubiquitin-specific protease 7 (USP7), death domain-associated protein (DAXX), murine double minute 2 (MDM2), murine double minute 4 (MDMX/ MDM4), p53, to explore the anti-tumor mechanism of nutlin-3; co-immunoprecipitation (Co-IP) assay was used to detect the effect of nutlin-3 on the interaction between MDM2, MDMX and p53. The results showed that nutlin-3 inhibited the proliferation of H460 in a time- and concentration-dependent manner. The results of cell cycle and apoptosis showed that nutlin-3 could block the H460 cell cycle in the G0/G1 phase, and induce apoptosis by activating cleaved-PARP. Western blot results showed that nutlin-3 could up-regulate the expression of USP7, DAXX, MDM2, MDMX and p53 in H460 cells. Co-IP results showed that nutlin-3 inhibited the protein interactions between MDM2 and p53 and MDM2 and MDMX. In conclusion, nutlin-3 can significantly inhibit the proliferation of wild-type p53 cancer cells and induce cell cycle arrest and apoptosis, which may be related to the disruption of MDM2/MDMX's interaction with p53 to activate the MDM2-p53 signaling pathway.

Oral probiotics are susceptible to the gastrointestinal environment, so the number of probiotics reaching the intestine is small and difficult to colonize, limiting the application of probiotic therapy. In this study, Lactobacillus rhamnosus (LGG), a common probiotic, was chosen as a model, and layer-by-layer encapsulated LGG-loaded porous microspheres with glycol chitosan (GCS) and sodium alginate (SA) were prepared to investigate it's in vitro properties. Poly-L-lactic acid porous microspheres (PLPM) were prepared by the complex milk-solvent evaporation method, with rounded morphology, uniform size, open and connected porous structure, and the average particle size of 138.5 μm. The PLPM were co-incubated with LGG for 8 h at 37 ℃ to obtain the LGG-loaded porous microspheres (LPM) with high bacterial loadings. The surface of the LPM were wrapped with GCS and SA layer by layer by electrostatic action to obtain the layer-by-layer encapsulated LGG-loaded porous microspheres with GCS and SA (AGLPM). In vitro experiments demonstrated that AGLPM could tolerate simulated gastric fluid at pH 1.2 and simulated intestinal fluid at pH 7.4 for 2 h, and its stability was significantly better than that of bare LGG. AGLPM was a better probiotic dosage form.

Plasma exosomes (Pla-Exos) were extracted from rats by ultracentrifugation. The ultracentrifugation method extracted rat Pla-Exos at a speed of 150 000 ×g for 2.5 h. Various purification methods including ultracentrifugation, magnetic bead capture method, and ultrafiltration would be employed to purify Pla-Exos. The Pue-Exos were prepared via sonication-assisted and co-incubation methods. The influence factors and levels of the preparation of plasma exosomes carrying puerarin (Pue-Exos) were investigated by RSM plus CCD method with encapsulation rate as an index. Then the characterization, the stability, and in vitro release of Pue-Exos were determined. The particle sizes of exosomes purified by ultracentrifugation, ultrafiltration, or magnetic bead capture method were all within the range of 30-150 nm. The Western blot results showed that purified Pla-Exos and Pue-Exos contained marker proteins TSG101, CD63, and CD81. The TEM showed that purified Pla-Exos and Pue-Exos exhibited well-defined double-layered membrane vesicle structures. The optimal prescription conditions for preparing Pue-Exos were finally determined as follows: 1 h ultrasound time, 39 W ultrasound power, mass ratio of 10∶1. The prepared Pue-Exos exhibit good stability and sustained release, significantly enhancing the in vitro transpermeability of puerarin across the blood-brain barrier (P < 0.01). This study was approved by the Experimental Animal Ethics Review Committee of Hebei North University, and ethics approval number was HBNV202307012103.

Current physical and chemical dosimeters are limited in that they cannot directly measure the biological effects of radiation or detect it within the body. Biosensors based on engineered probiotics demonstrate high stability and safety, can be used to detect ionizing radiation in vivo. In this study, an oral engineered microbial sensor for ionizing radiation detection has been developed. The Escherichia coli Nissle 1917 (EcN) was selected as the chassis strain. Using CRISPR/Cas9 gene-editing technology, the cryptic plasmids of EcN were successfully removed to yield the chassis strain ΔEcN. To design a radiation-responsive gene circuit, the recA promoter from the SOS response pathway was utilized as the radiation response element, while a fluorescent protein served as the reporter element. This system was designed to be induced by mitomycin C and γ-rays. The performance of engineered bacteria with various gene circuits was characterized and optimized, resulting in the selection of improved candidate strains. Animal experiments were approved by the Ethics Committee of Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (approval number: IACUC-DWZX-2022-521). After comparing the in vivo radiation detection capabilities of these strains, the EC-8, which exhibited higher sensitivity, was identified as the final oral microbial sensor. This research applies synthetic biology principles to design and engineer a probiotic capable of detecting ionizing radiation within the body. The findings offer a novel method for in vivo ionizing radiation detection and lay the foundation for the development of live biotherapeutics for the precise diagnosis of radiation damage.

High-altitude sleep disturbance is a common acute high-altitude disease that can trigger physiological discomfort such as acute high-altitude reactions, with a lack of safe and effective preventive medications in clinical practice. Based on the gut-brain axis theory, this study designed and prepared a synbiotics combining Lactobacillus rhamnosus (LGG)-Lycium barbarum polysaccharide (LBP). First, the LGG-LBP synbiotics was prepared and evaluated. The mice were randomly divided into healthy, model, positive control (acetazolamide), LBP, LGG and LGG-LBP synbiotics group. After 7 days of administration, the mouse model of high-altitude sleep disturbance was established, the treating effects were evaluated through sleep duration, behavioral, hemogram test, and the content of tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) in blood. 16S rRNA sequencing was used to analyze the changes of gut microbiota, and the pathological changes of small intestine were observed. The LGG-LBP synbiotics prolonged sleep duration, improved exploratory ability and short-term memory, promoted blood cell recovery. Moreover, LGG-LBP synbiotics enhanced the abundance of probiotics in the gut, and reduced intestinal inflammation. LGG-LBP synbiotics may be a potential prophylactic drug for high-altitude sleep disturbance. The animal operation was approved by the Ethics Committee of the Academy of Military Medical Sciences, Academy of Military Science (Approval number: IACUC-DWZX-2022-511). All experiments were conducted in accordance with relevant guidelines and regulations.

Radiation enteritis (RE) is the most common complication during radiotherapy, although safe and effective drugs for the treatment of RE are deficient. Probiotics have been demonstrated to own antiradiation function. Synbiotics are composed of probiotics and prebiotics, which enhance the ability of probiotics. Here, inulin gel (IG) that own the ability to resist gastric acid and retain in the colon were used to load three types of probiotics, including Bacillus cereus (BC), Bacillus licheniformis (BL), and Lactobacillus reuteri (LR), respectively. The RE pharmacodynamic studies of them were conducted. The probiotics were embedded in the IG by scanning electron microscopy and confocal laser scanning microscopy. IG promoted the growth of probiotics compared to probiotics alone. Animal experiments were approved by the Ethics Committee of the Academy of Military Medical Sciences and the experiments were conducted in accordance with relevant guidelines and regulations (No. IACUC-DWZX-2024-P510). Mice experienced the whole abdominal irradiation of 13 Gy γ rays to form RE models. Compared to correspondingly probiotics alone, all probiotic-loaded IG synbiotics had the better prevention and treatment efficiencies in repairing the intestinal barriers, improving the structure of crypts, oxidative stress, inflammation, and imbalanced gut microbiota and promoting the recovery of intestinal villus, where the Bacillus cereus-loaded IG was the best. This study provides a novel therapeutic for the prevention and treatment of RE.

The 95% ethanol extract of Curcuma longa was isolated and purified by silica gel column chromatography, polyamide column chromatography, preparative thin layer chromatography and semi-preparative HPLC. Then, the structures of the obtained compounds were identified by HR-MS, IR, and NMR. Absolute configuration of the new compound was determined by calculating ECD. Finally, two bisabolane-type sesquiterpenoids were obtained from C. longa and identified as (7S)-1,3,5,10-bisabolatetraen-3-nitro-9-one (1) and turmeronol A (2). Compound 1 was a novel nitro-substituted bisabolane-type sesquiterpenoid.

Pulmonary fibrosis (PF) is a lung disease with a very poor prognosis that seriously affects the quality of life of patients and is characterized by scarring and thickening of the tissue surrounding the alveolar walls, ultimately leading to respiratory failure. Currently, the Food and Drug Administration (FDA) approved drugs for the treatment of PF include pirfenidone and nidazanib, however, these two drugs can only delay the progression of the disease but cannot achieve the reversal of PF, and their clinical application is limited due to high price and multiple adverse effects. The pathogenesis of PF has not been fully elucidated, and studies have demonstrated that aberrant immune cell activation and regulation play an important role in PF. This review aims to discuss the role of immune cell activation and regulation in PF in recent years. The aim of this review is to discuss recent advances in the study of the role of immune cells in the process of PF, with the aim of providing theoretical guidance for the development of novel immunotherapies.