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.

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.

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.

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.

In the early stage of the project, it was found that natural pentacyclic triterpenes liquidambaric acid regulates the NEDD8 modification of Cullin2. This study aims to find more triterpenoid natural active molecules targeting Cullin family members and reveal its mechanism of action. Western blot was used to detect natural products that can significantly change the total protein NEDD8 modification and specific Cullin protein NEDD8 modification in cells; microscale thermophoresis (MST) was used to detect the direct binding of candidate small molecule oleanonic acid to TRAF family proteins, and the binding at the level of living cells was verified by cellular thermal shift assay (CETSA). Proximity ligation assay (PLA) was used to investigate the regulatory effect of oleanonic acid on the protein interaction between TNF receptor-associated factor 1 (TRAF1) and Cullin1 NEDD8 modified complex. Three pentacyclic triterpenoids were found to significantly inhibit NEDD8 modification in cells, among which oleanonic acid had the strongest effect on blocking NEDD8 modification. Different from the previous identification that liquidambaric acid regulates Cullin2/5, oleanonic acid can also specifically induce NEDD8-modified Cullin1 to transform into its unmodified form. And binding experiments showed that oleanonic acid could directly bind to TRAF1 at the level of cell lysate and living cells. Further mechanism studies found that oleanonic acid significantly changed the protein interaction between TRAF1 and Cullin1 NEDD8 modified complex. The above results indicate that oleanonic acid targets TRAF1 and regulates its interaction with NEDD8 modification complex to inhibit NEDD8 modification of Cullin.

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.

The study aims to provide an important basis for IMH020 production process and quality control and develop an HPLC method for the determination of related substances in IMH020. The analysis was performed on a Waters Symmetry C₁₈ column (250 mm × 4.6 mm, 5 µm). The mobile phase A was acetonitrile and the mobile phase B was water-0.2% glacial acetic acid solution with isocratic elution. The flow rate was 1.0 mL·min^-1 and the column temperature was maintained at 30 ℃. The wavelength was 270 and 300 nm. The injection volume was 10 μL. The results show that all the related substances gained a completely chromatographic separation. Good linear relationships of all the related substances (I-1, I-2, I-3a, I-3b, I-4 and I-5) were obtained (r ≥ 0.998 9) and recoveries were in the range of 93.1%-97.8% (RSD ≤ 2%, n = 9). Only I-3a and I-3b were detected below the limit for specified impurities in the three batches of samples, and the largest unspecified single impurity and the total impurities were within the limit. The HPLC method established in this paper is simple, sensitive and accurate, and can be used for the determination of related substances of IMH020.

Whole body irradiation (WBI) injury is defined multi-organ damages caused by whole-body exposure to ionizing radiation. The traditional radioprotective drug, amifostine, has significant adverse effects. Probiotics are reported to have radioprotective function, although their therapeutic efficacy is low due to poor gastrointestinal tolerance and the insufficient retention and colonization in the colon. In this study, chitosan/tannic acid double-layer-coated Lactobacillus reuteri was prepared, which was encapsulated in calcium alginate hydrogel microspheres to get an engineered probiotic-loaded microsphere formulation. The bilayer coating was confirmed by twice inversions of zeta potentials. Moreover, the coating improved bacterial adhesion and aggregation. Optical microscopy revealed the smooth morphology of microspheres, laser confocal imaging showed the uniform distribution of coated bacteria in microspheres, and scanning electron microscopy exhibited pores in the surface. The microspheres exhibited in vitro gastrointestinal resistance with rapidly swelling in the colonic environment to release bacteria. All the animal experiments were approved by Academy of Military Medicine Sciences (Approval No: IACUC-DWZX-2024-P510) and conducted in compliance with relevant guidelines. The 6.5 Gy whole-body irradiated mouse model was established. Starting from 2 days prior to irradiation, probiotic-loaded microspheres were administered via oral gavage consecutively for 6 days. Compared with the model group, the bacteria-loaded microspheres demonstrated protective effects on the hematopoietic system by promoting the recovery of red blood cells and platelets, maintaining the morphology of splenic red pulp and white pulp, and preserving bone marrow nucleated cells along with their proliferative capacity. Engineered probiotics have expanded the spectrum of radioprotective drugs, offering novel insights for the development of live biotherapeutic products aimed at preventing and treating radiation-induced injury.

The methanol extract of Pteris wallichiana was separated and purified by MCI gel, sephadex LH 20, flash C18 and silica gel column chromatography combined with semi-pre HPLC. The chemical structures of the isolated compounds were identified by MS, IR, NMR, etc. Five sesquiterpene compounds were isolated from Pteris wallichiana and identified as 6,7-tetrahydrofuran-(2S, 3S)-pterosin C-3-O-β-D-(6′-acetyl)-Glu (1), (2S, 3S)-pterosin C-3-O-β-D-Glu (2), (2S)-pterosin A (3) and (2S)-13-hydroxyl-pterosin A (4), (2R, 3S)-2-hydroxyl-pterosin C (5). Compound 1 is a new sesquiterpene, compounds 3-5 were isolated for the first time. In vitro bioactivity assay showed that compound 1 was able to inhibit the proliferation of 4T1 and EMT6 cells, and possessed significant anti-triple-negative breast cancer bioactivity.

Four terpenoids were isolated from the neutral portion of petroleum ether extract of Artemisia annua by several chromatographic methods, such as silica gel, MCI Gel CHP-20, ODS, Sephadex LH-20 and semi-preparative HPLC. Their structures were identified by HR-MS and nuclear magnetic resonance spectroscopy. These compounds were defined as (1S,4S,5R,6S,9R,10R)-4-ethoxy-9,10-dimethyloctahydrofuro-(3,2-i)-isochromen-11(4H)-one (1), 3a,4,5,6,6a,7-hexahydro-3,6-dimethyl-9-methyl-2H-naphtho[8a,1-b]furan-2,8(3H)-dione (2), kobusone (3) and 1,2-campholide (4). Compound 1 is a new compound, of which the absolute configuration was established by single crystal X-ray crystallographic analysis. Compound 2 is a new natural product. Compounds 3 and 4 are first isolated from the Artemisia genus.