Tong-Cun Zhang

The manufacturing process contributes significantly to the proliferation, metabolic state, and functional persistence of chimeric antigen receptor (CAR)-T cells. However, how different culture systems regulate CAR-T cell metabolism and thereby influence their long-term antitumor activity remains poorly understood. In this study, we compared dynamic cultivation using a wave bioreactor with static expansion systems (gas-permeable and conventional T-flasks) for the production of CD99-specific CAR-T cells. CAR-T cells expanded by the wave bioreactor exhibited faster proliferation and stronger cytotoxicity during culture. Upon repeated antigen stimulation, they retained these enhanced functional properties and showed the reduced expression of immune checkpoint molecules, preferentially preserved memory-like subsets, and displayed transcriptional features consistent with memory maintenance and exhaustion resistance. Targeted metabolomic profiling revealed enhanced Tricarboxylic Acid (TCA) cycle activity and features consistent with sustained oxidative phosphorylation, supporting mitochondrial-centered metabolic reprogramming. In a Ewing sarcoma xenograft model, wave bioreactor-cultured CAR-T cells showed a greater percentage of memory-like tumor-infiltrating lymphocytes. Collectively, these results indicate that wave bioreactor-based dynamic cultivation promotes mitochondrial metabolic reprogramming, which is characterized by an enhanced TCA cycle and sustained oxidative phosphorylation, thereby sustaining CAR-T cell functionality and providing a robust platform for the manufacturing of potent and durable cellular therapeutics.

Artemisia frigida Willd. (A. frigida), a traditional medicinal herb widely distributed in northern China, Mongolia, and Siberia, has garnered increasing scientific interest due to its diverse phytochemical profile and extensive pharmacological potential. Modern studies have identified a wide range of bioactive compounds in A. frigida, including flavonoids, sesquiterpene lactones and phenolic acids. These compounds exhibit various biological activities, such as antioxidant, anti-inflammatory, anticancer, and antimicrobial effects. This review systematically summarizes the research progress on the chemical constituents of A. frigida and their extraction and separation methods, including solvent extraction, ultrasonic-assisted extraction, macroporous resin adsorption, and chromatography-based techniques. By integrating traditional knowledge with modern pharmacological evidence, this review provides a scientific foundation for the further development and utilization of A. frigida in functional food, pharmaceuticals, and ethnomedicine.

Background: Chimeric antigen receptor T cell (CAR-T) therapy targeting cluster of differentiation 19 (CD19) has shown promise in treating B-cell acute lymphoblastic leukemia (B-ALL), but the long-term efficacy of CD19-targeted CAR-T therapy remains limited. Programmed cell death protein 1 (PD-1), an inhibitory receptor on activated T cells, engages programmed death ligand 1 (PD-L1) in the tumor microenvironment, suppressing CAR-T cell function by impairing key signaling pathways. To address this limitation, we investigated the effect of short hairpin RNA (shRNA)-mediated PD-1 knockdown on alleviating CAR-T cell exhaustion, preserving memory phenotype, and enhancing the long-term efficacy of CD19-targeted CAR-T cells. Methods: To overcome this limitation, we employed shRNA to achieve PD-1 knockdown in CD19-targeting CAR-T cells. Following screening multiple shRNA candidates, we assessed the cytotoxicity, cytokine secretion, and proliferation of PD-1 knockdown CAR-T cells. Their memory phenotype, exhaustion marker, and cytolytic function were further evaluated after repeated antigen stimulation. Therapeutic efficacy and safety were validated in NOD.Cg-PrkdC^scid Il2rg^tm1/Vst (NPG) mice. Results: PD-1 knockdown significantly enhanced CAR-T cell cytotoxicity and persistence against PD-L1+ RAJI (human Burkitt’s lymphoma cell line) cells. After three rounds of antigen stimulation, PD-1 knockdown CAR-T cells sustained cytotoxic activity, retained a memory-like phenotype, and displayed attenuated exhaustion markers. In vivo, they prolonged survival without inducing cytokine release syndrome (CRS), indicating improved efficacy and safety. Conclusions: PD-1 knockdown mitigates tumor-induced T-cell exhaustion and represents a promising strategy to enhance the durability and potency of CAR-T cell therapy in cancer treatment.

Alcoholic liver disease is one of the diseases with a high mortality rate worldwide, resulting from excessive and chronic alcohol consumption. With the rapid rise of intestinal microbial research, more and more researchers have begun to focus on the role of probiotics in preventing, alleviating or treating diseases. In this study, effects of lactic acid bacteria (LAB), a general type of probiotic, isolated from the fermented grains of Chinese baijiu, on alcohol-induced liver injury and alcohol metabolism were investigated, and the results showed that Lactiplantibacillus pentosus LTJ12, Pediococcus acidilactici LTJ28, Lactiplantibacillus plantarum LTJ30, and Pediococcus acidilactici LTJ32 could prevent drunkenness and sober up, and had a good protective effect on alcoholic liver injury. These LAB, especially Lactiplantibacillus, can reduce the drunken rate and mortality of drinking mice, shorten the sobriety time, decrease the content of ethanol in serum, reduce the activity or content of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), triglyceride (TG) and malondialdehyde (MDA), increase the activity or content of superoxide dismutase (SOD), glutathione (GSH) and nitric oxide (NO), and also improve the activity of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) of the liver. The LAB intervention basically reversed the changes in the mRNA levels of genes related to ethanol/drug metabolism (CYP2E1, ADH1 and ALDH2), antioxidant markers (SOD2 and CAT), cellular inflammation, apoptosis and proliferation (SIRT1, SMYD3 and BRD4) and lipid metabolism (SREBP1 and FASn). In addition, we have found that the regulatory effect of these probiotics may be related to the SMYD3/BRD4 pathway, which needs further detailed research. Besides, the probiotics increased the abundance of gut microbes, restored the imbalance of the intestinal flora caused by alcohol consumption, and regulated the changes in the intestinal short-chain fatty acid content caused by chronic alcohol consumption. These results suggested that these baijiu-derived probiotics can effectively prevent drunkenness and chronic alcoholic liver injury. It is of great significance to provide scientific basis for subsequent research and development of new anti-alcoholism health products based on probiotics and the intestinal microecological regulation mechanism.

This study investigated the effect of astragalus polysaccharide (APS, an ingredient with hypoglycemic function in a traditional Chinese herbal medicine) on gut microbiota and metabolites of type 2 diabetes mellitus (T2DM) patients using a simulated fermentation model in vitro. The main components of APS were isolated, purified, and structure characterized. APS fermentation was found to increase the abundance of Lactobacillus and Bifidobacterium and decrease the Escherichia-Shigella level in the fecal microbiota of T2DM patients. Apart from increasing propionic acid, APS also caused an increase in all-trans-retinoic acid and thiamine (both have antioxidant properties), with their enrichment in the KEGG pathway associated with thiamine metabolism, etc. Notably, APS could also enhance fecal antioxidant properties. Correlation analysis confirmed a significant positive correlation of Lactobacillus with thiamine and DPPH-clearance rate, suggesting the antioxidant activity of APS was related to its ability to enrich some specific bacteria and upregulate their metabolites.

Mesenchymal stem cells (MSCs) can be used as a cell source for cultivated meat production due to their adipose differentiation potential, but MSCs lose their stemness and undergo replicative senescence during expansion in vitro. Autophagy is an important mechanism for senescent cells to remove toxic substances. However, the role of autophagy in the replicative senescence of MSCs is controversial. Here, we evaluated the changes in autophagy in porcine MSCs (pMSCs) during long-term culture in vitro and identified a natural phytochemical, ginsenoside Rg2, that could stimulate pMSC proliferation. First, some typical senescence characteristics were observed in aged pMSCs, including decreased EdU-positive cells, increased senescence-associated beta-galactosidase activity, declined stemness-associated marker OCT4 expression, and enhanced P53 expression. Importantly, autophagic flux was impaired in aged pMSCs, suggesting deficient substrate clearance in aged pMSCs. Rg2 was found to promote the proliferation of pMSCs using MTT assay and EdU staining. In addition, Rg2 inhibited D-galactose-induced senescence and oxidative stress in pMSCs. Rg2 increased autophagic activity via the AMPK signaling pathway. Furthermore, long-term culture with Rg2 promoted the proliferation, inhibited the replicative senescence, and maintained the stemness of pMSCs. These results provide a potential strategy for porcine MSC expansion in vitro.

Fumonisin (FB) is one of the most common mycotoxins contaminating feed and food, causing severe public health threat to human and animals worldwide. Until now, only several transaminases were found to reduce FB toxicity, thus, more fumonisin detoxification transaminases with excellent catalytic properties required urgent exploration for complex application conditions. Herein, through gene mining and enzymatic characterization, three novel fumonisin detoxification transaminases—FumTSTA, FumUPTA, FumPHTA—were identified, sharing only 61–74% sequence identity with reported fumonisin detoxification transaminases. Moreover, the recombinant proteins shared diverse pH reaction ranges, good pH stability and thermostability, and the recombinant protein yields were also improved by condition optimum. Furthermore, the final products were analyzed by liquid chromatography-mass spectrometry. This study provides ideal candidates for fumonisin detoxification and meets diverse required demands in food and feed industries.

Lactic acid bacteria have received a significant amount of attention due to their probiotic characteristics. The species Lactiplantibacillus plantarum and Lactiplantibacillus pentosus are genotypically closely related, and their phenotypes are so similar that they are easily confused and mistaken. In the previous study, an ethanol-resistant strain, LTJ12, isolated from the fermented grains of soy sauce aroma type baijiu in North China, was originally identified as L. plantarum through a 16S rRNA sequence analysis. Here, the genome of strain LTJ12 was further sequenced using PacBio and Illumina sequencing technology to obtain a better understanding of the metabolic pathway underlying its resistance to ethanol stress. The results showed that the genome of strain LTJ12 was composed of one circular chromosome and three circular plasmids. The genome size is 3,512,307 bp with a GC content of 46.37%, and the number of predicted coding genes is 3248. Moreover, by comparing the coding genes with the GO (Gene Ontology), COG (Cluster of Orthologous Groups) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases, the functional annotation of the genome and an assessment of the metabolic pathways were performed, with the results showing that strain LTJ12 has multiple genes that may be related to alcohol metabolism and probiotic-related genes. Antibiotic resistance gene analysis showed that there were few potential safety hazards. Further, after conducting the comparative genomics analysis, it was found that strain LTJ12 is L. pentosus but not L. plantarum, but it has more functional genes than other L. pentosus strains that are mainly related to carbohydrate transport and metabolism, transcription, replication, recombination and repair, signal transduction mechanisms, defense mechanisms and cell wall/membrane/envelope biogenesis. These unique functional genes, such as gene 2754 (encodes alcohol dehydrogenase), gene 3093 (encodes gamma-D-glutamyl-meso-diaminopimelate peptidase) and some others may enhance the ethanol tolerance and alcohol metabolism of the strain. Taken together, L. pentosus LTJ12 might be a potentially safe probiotic with a high ethanol tolerance and alcohol metabolism. The findings of this study will also shed light on the accurate identification and rational application of the Lactiplantibacillus species.

Preservation and transportation are essential for the clinical application of chimeric antigen receptor T (CAR-T) cells. This study aimed to optimize a cryopreservation solution for CAR-T cells and evaluate the antitumor efficiency of CAR-T cells using this optimized solution in vitro and in vivo. First, the stability of the cryopreservation solution for CAR-T infusion was detected by the L27 (3⁷) orthogonal experiment. Subsequently, osmolality and pH were analyzed for the preservation reagent. Additionally, apoptosis and CAR expression of CAR-T cells were measured by flow cytometry, and the cytotoxicity was determined by calcein-AM staining. The results showed that cryopreservation solutions used in this study demonstrated high chemical stability, which induced only 2% CAR-T cells apoptosis in optimal solutions, which were slightly lower than other commercial solutions. Moreover, the CAR expression was not significantly affected by preservation with these solutions. There were no significant differences in the cytotoxicity between fresh and thawed CAR-T cells cryopreserved in the cryopreservation solutions in vivo and in vitro. This study developed a new cryopreservation solution for CAR-T cells, and it was safe and also had negligible effects on the CAR-T cells antitumor activity.

This study investigated the influence of chitosan oligosaccharides (COSs) on a thioacetamide-induced hepatic encephalopathy (HE) Wistar rat model. COS treatment statistically reduced the false neurotransmitters and blood ammonia in HE rats, along with the suppression of oxidative stress and inflammation. The disbalanced gut microbiota was detected in HE rats by 16S rDNA sequencing, but the abundance alterations of some intestinal bacteria at either the phylum or genus level were at least partly restored by COS treatment. According to metabolomics analysis of rat feces, six metabolism pathways with the greatest response to HE were screened, several of which were remarkably reversed by COS. The altered metabolites might serve as a bridge for the alleviated HE rats treated with COS and the enhanced intestinal bacterial structure. This study provides novel guidance to develop novel food or dietary supplements to improve HE diseases due to the potential beneficial effect of COS on gut-liver axis.