BACKGROUND:Macrophage migration inhibitory factor(MIF)is a pleiotropic cytokine,which is secreted in different types of stem cells and can regulate the proliferation,differentiation and migration of various types of stem cells.Our previous research has confirmed that human embryonic stem cells secrete MIF and that its concentration in the culture medium is relatively stable.However,whether MIF is involved in the survival,proliferation and differentiation of human embryonic stem cells remains unclear. OBJECTIVE:To investigate the effects of MIF on survival,proliferation,and differentiation of human embryonic stem cells. METHODS:(1)Human embryonic stem cells H9 were cultured.The growth curve of cells was detected and plotted by CCK-8 assay.Enzyme-linked immunosorbent assay was used to determine the level of MIF in the medium.(2)To determine the effects of exogenous MIF on the survival and proliferation of human embryonic stem cells,different groups were established:the control group,which was cultured in stem cell medium without any modifications;the exogenous MIF group,which was treated with different concentrations(30,100,300 ng/mL)of MIF in the stem cell medium;the MIF inhibitor ISO-1 group,which was treated with different concentrations(2,7,21 μmol/L)of ISO-1 in the stem cell medium;and the MIF+ISO-1 group,which was treated with different concentrations of ISO-1 along with 100 ng/mL of MIF.Cell viability was assessed using the CCK-8 assay.(3)To further elucidate the effect of MIF gene on survival and proliferation of human embryonic stem cell,the MIF knockout H9 cell line was constructed by CRISPR-Cas 9 technology to observe the lineage establishment.(4)To determine the effect of high concentrations of MIF on human embryonic stem cell differentiation,100 ng/mL MIF and 100 ng/mL of CXCR4 neutralizing antibody were separately added to the normal stem cell culture medium.The expression levels of self-renewal factors(KLF4,c-MYC,NANOG,OCT4,and SOX2)and differentiation transcription factors(FOXA2,OTX2)were measured using real-time quantitative polymerase chain reaction,immunofluorescence staining,and western blot analysis. RESULTS AND CONCLUSION:(1)The logarithmic growth phase of H9 cells was between 3-6 days.Under normal growth conditions,human embryonic stem cells secreted MIF at a concentration of approximately 20 ng/mL,independent of cell quantity.(2)Compared to the control group,the addition of different concentrations of MIF had no effect on the proliferation of human embryonic stem cells(P>0.05).ISO-1 significantly inhibited the proliferation of human embryonic stem cells,with a stronger inhibition observed at higher concentrations of ISO-1(P<0.05).The addition of MIF in the presence of ISO-1 reduced the inhibitory effect of ISO-1(P<0.05).(3)Real-time quantitative polymerase chain reaction showed that knocking out 50%of the MIF gene resulted in a significant decrease in the growth vitality of human embryonic stem cells and failure to establish cell lines.(4)Adding 100 ng/mL exogenous MIF to the culture medium resulted in a decrease in the mRNA,protein,and fluorescence expression levels of the self-renewal transcription factor KLF4,while the mRNA,protein,and fluorescence expression levels of the differentiation factor FOXA2 increased.(5)When 100 ng/mL CXCR4 neutralizing antibody was added to the culture medium,the mRNA and protein expression levels of KLF4 increased,while the mRNA and protein expression levels of FOXA2 decreased,contrary to the expression trend observed in the MIF group.In conclusion,the endogenous secretion of MIF by human embryonic stem cells is essential for their survival.The addition of MIF to the culture medium does not promote the proliferation of human embryonic stem cells.However,it can lead to a decrease in the expression of the self-renewal factor KLF4 and an increase in the expression of the transcription factor FOXA2.This provides a clue for further investigation into the effects and mechanisms of MIF on the differentiation of human embryonic stem cells.The MIF-CXCR4 axis plays a regulatory role in this process.

ObjectiveTo explore the possible mechanism of Modified Gegen Qinlian Decoction （加味葛根芩连汤， MGQD） in the treatment of ulcerative colitis （UC） based on intestinal mucus barrier. MethodsThirty C57BL/6 mice were randomly divided into a control group， a model group and a MGQD group with 10 mice in each. Dextran Sulfate Sodium Salt （DSS） was used to construct the UC model in all groups except for the control group. Meanwhile， mice in the MGQD group were given 20 g/kg of MGQD decoction by gavage according to their body weight， while those in the control group and model group were given 0.2 ml/20 g of pure water by gavage， once a day for 7 consecutive days. On the day following the last gavage， the body weight， disease activity index （DAI） score， spleen weight， and colon length were compared. The pathological changes of the intestinal mucosal tissues were observed by HE staining； the protein expression levels of mucin 2 （MUC2） and leucine-rich repeat G protein-coupled receptor 5 （Lgr5） in the intestinal mucosal tissues were detected by immunofluorescence； the cuprocytes in the intestinal mucosal tissues were detected by AB/PAS staining； and the expression level of Ki67 in the intestinal mucosal tissues was detected by immunohistochemistry. ResultsHE staining showed that the colon mucosal tissue of the mice in the control group was intact. In the model group， the colon mucosal epithelial structure was severely damaged， with a large amount of inflammatory cell infiltration in the mucosal propria. In the MGQD group， the mucosal tissue structure was partially lost， with a small amount of inflammatory cell infiltration.The body weight and colon length of mice in the model group decreased significantly compared to those in the control group， while DAI scores and spleen weight increased， and the levels of MUC2， Ki67， Lgr5 proteins， and the number of goblet cells were significantly reduced （P＜0.01）. Compared to the model group， the MGQD group had increased body weight of mice， colon length， and decreased DAI scores and spleen weight； the levels of MUC2， Ki67， Lgr5 proteins， and the number of goblet cells were increased （P＜0.05 or P＜0.01）. ConclusionMGQD has a favorable ameliorative effect on UC-related symptoms and pathological tissue damage， and its mechanism of action may be related to the restoration of the prolife-ration and differentiation of intestinal stem cells into goblet cells， thereby promoting the repair of the intestinal mucus barrier.

BACKGROUND: Several studies have demonstrated the occurrence of secondary tumors as a rare but significant complication of chimeric antigen receptor T (CAR-T) cell therapy, underscoring the need for a detailed investigation. Given the limited variety of secondary tumor types reported to date, a comprehensive characterization of the various secondary tumors arising after CAR-T therapy is essential to understand the associated risks and to define the role of the immune microenvironment in malignant transformation. This study aims to characterize the immune microenvironment of a newly identified secondary tumor post-CAR-T therapy, to clarify its pathogenesis and potential therapeutic targets. METHODS: In this study, the bone marrow (BM) samples were collected by aspiration from the primary and secondary tumors before and after CD19 CAR-T treatment. The CD45 + BM cells were enriched with human CD45 microbeads. The CD45 + cells were then sent for 10× genomics single-cell RNA sequencing (scRNA-seq) to identify cell populations. The Cell Ranger pipeline and CellChat were used for detailed analysis. RESULTS: In this study, a rare type of secondary chronic myelomonocytic leukemia (CMML) were reported in a patient with diffuse large B-cell lymphoma (DLBCL) who had previously received CD19 CAR-T therapy. The scRNA-seq analysis revealed increased inflammatory cytokines, chemokines, and an immunosuppressive state of monocytes/macrophages, which may impair cytotoxic activity in both T and natural killer (NK) cells in secondary CMML before treatment. In contrast, their cytotoxicity was restored in secondary CMML after treatment. CONCLUSIONS This finding delineates a previously unrecognized type of secondary tumor, CMML, after CAR-T therapy and provide a framework for defining the immune microenvironment of secondary tumor occurrence after CAR-T therapy. In addition, the results provide a rationale for targeting macrophages to improve treatment strategies for CMML treatment.
Humans ; Lymphoma, Large B-Cell, Diffuse/therapy* ; Tumor Microenvironment/genetics* ; Antigens, CD19/metabolism* ; Leukemia, Myelomonocytic, Chronic/genetics* ; Immunotherapy, Adoptive/adverse effects* ; Male ; Single-Cell Analysis/methods* ; Female ; Sequence Analysis, RNA/methods* ; Receptors, Chimeric Antigen ; Middle Aged

Osteosarcoma (OS), chondrosarcoma (CS), and Ewing sarcoma (ES) represent primary malignant bone tumors and pose significant challenges in oncology research and clinical management. Conventional research methods, such as two-dimensional (2D) cultured tumor cells and animal models, have limitations in recapitulating the complex tumor microenvironment (TME) and often fail to translate into effective clinical treatments. The advancement of three-dimensional (3D) culture technology has revolutionized the field by enabling the development of in vitro constructed bone tumor models that closely mimic the in vivo TME. These models provide powerful tools for investigating tumor biology, assessing therapeutic responses, and advancing personalized medicine. This comprehensive review summarizes the recent advancements in research on 3D tumor models constructed in vitro for OS, CS, and ES. We discuss the various techniques employed in model construction, their applications, and the challenges and future directions in this field. The integration of advanced technologies and the incorporation of additional cell types hold promise for the development of more sophisticated and physiologically relevant models. As research in this field continues to evolve, we anticipate that these models will play an increasingly crucial role in unraveling the complexities of malignant bone tumors and accelerating the development of novel therapeutic strategies.
Bone Neoplasms/pathology* ; Humans ; Osteosarcoma/pathology* ; Tumor Microenvironment ; Sarcoma, Ewing/pathology* ; Chondrosarcoma/pathology* ; Animals ; Cell Culture Techniques/methods* ; Cell Culture Techniques, Three Dimensional/methods* ; Cell Line, Tumor

The prevalence of Class III malocclusion varies among different countries and regions. The populations from Southeast Asian countries (Chinese and Malaysian) showed the highest prevalence rate of 15.8%, which can seriously affect oral function, facial appearance, and mental health. As anterior crossbite tends to worsen with growth, early orthodontic treatment can harness growth potential to normalize maxillofacial development or reduce skeletal malformation severity, thereby reducing the difficulty and shortening the treatment cycle of later-stage treatment. This is beneficial for the physical and mental growth of children. Therefore, early orthodontic treatment for Class III malocclusion is particularly important. Determining the optimal timing for early orthodontic treatment requires a comprehensive assessment of clinical manifestations, dental age, and skeletal age, and can lead to better results with less effort. Currently, standardized treatment guidelines for early orthodontic treatment of Class III malocclusion are lacking. This review provides a comprehensive summary of the etiology, clinical manifestations, classification, and early orthodontic techniques for Class III malocclusion, along with systematic discussions on selecting early treatment plans. The purpose of this expert consensus is to standardize clinical practices and improve the treatment outcomes of Class III malocclusion through early orthodontic treatment.
Humans ; Malocclusion, Angle Class III/classification* ; Orthodontics, Corrective/methods* ; Consensus ; Child

Benzylisoquinoline alkaloids (BIAs) are a structurally diverse group of plant metabolites renowned for their pharmacological properties. However, sustainable sources for these compounds remain limited. Consequently, researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches. CYP80B, a family of cytochrome P450 (CYP450) enzymes, plays a crucial role in BIA biosynthesis. Previously reported CYP80Bs are known to catalyze the 3'-hydroxylation of (S)-N-methylcoclaurine, with the N-methyl group essential for catalytic activity. In this study, we successfully cloned a full-length CYP80B gene (StCYP80B) from Stephania tetrandra (S. tetrandra) and identified its function using a yeast heterologous expression system. Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that StCYP80B could catalyze N-methylcoclaurine and coclaurine into their respective 3'-hydroxylated products. Notably, StCYP80B exhibited an expanded substrate selectivity compared to previously reported wild-type CYP80Bs, as it did not require an N-methyl group for hydroxylase activity. Furthermore, StCYP80B displayed a clear preference for the (S)-configuration. Co-expression of StCYP80B with the CYP450 reductases (CPRs, StCPR1, and StCPR2), also cloned from S. tetrandra, significantly enhanced the catalytic activity towards (S)-coclaurine. Site-directed mutagenesis of StCYP80B revealed that the residue H205 is crucial for coclaurine catalysis. Additionally, StCYP80B exhibited tissue-specific expression in plants. This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
Cytochrome P-450 Enzyme System/chemistry* ; Benzylisoquinolines/chemistry* ; Hydroxylation ; Plant Proteins/chemistry* ; Alkaloids/metabolism* ; Stephania tetrandra/genetics*

OBJECTIVES: To propose a hypothesis that aloe-emodin may inhibit scar tissue fibrosis through thrombospondin-1(THBS1)-PI3K-Akt pathway. METHODS: By cultivating fibroblasts derived from scar tissue after cleft palate surgery in humans, aloe emodin of different concentrations (10, 20, 30, 40 and 50 μmol/L) was added to the cells which activity was detected. At the same time, transcriptome sequencing was performed on scar tissue and cells, and bioinformatics methods were used to explore potential targets and signaling pathways of scar tissue fibrosis. RESULTS: Aloe-emodin had a concentration dependent inhibitory effect on fibroblast proliferation,with the 40 μmol/L concentration group showing the most significant effect. The results of tissue and cell sequencing indicated that differentially expressed genes were significantly enriched in extracellular matrix-receptor interaction pathway, and shared a common differential gene which was THBS1. The ORA analysis results indicated that differentially expressed genes, including THBS1, were significantly enriched in the PI3K-Akt signaling pathway. CONCLUSIONS Aloe emodin may inhibit the PI3K-Akt pathway by downregulating THBS1, thereby reducing the proliferation activity of fibroblasts derived from postoperative palatal scar tissue.
Thrombospondin 1/genetics* ; Humans ; Signal Transduction/drug effects* ; Fibroblasts/cytology* ; Proto-Oncogene Proteins c-akt/metabolism* ; Fibrosis ; Phosphatidylinositol 3-Kinases/metabolism* ; Cicatrix/metabolism* ; Cell Proliferation/drug effects* ; Anthraquinones/pharmacology* ; Cells, Cultured

ObjectiveTo assess the capacity of health emergency drills for poisoning emergencies at the municipal level in Guangdong Province. Methods A total of 21 municipal teams from cities in Guangdong Province participated in the health emergency drill competition, which included comprehensive tests and practical assessments. Results The pass rate for the total score, comprehensive tests, practical assessments of 21 municipal teams was 66.7%, 33.3%, 66.7%, respectively. The pass rate of the comprehensive tests was lower than that of practical assessments (P<0.01). The pass rate for the total score, comprehensive tests, and practical assessments of team from the Pearl River Delta region was higher than those in non-Pearl River Delta regions (88.9% vs 50.0%, 55.5% vs 16.7%, 88.9% vs 50.0%). For the four comprehensive test items, the highest pass rate was for personal protective principles against chemical poisoning (57.1%). For the five practical assessment items, the highest pass rate was for the selection and matching of personal protective equipment and practice of poisoning detection (both 71.4%). Conclusion It is urgent to improve the capacity of health emergency drills at the municipal level in Guangdong Province. Emphasis should be placed on strengthening capacity building in teams from non-Pearl River Delta regions.

ObjectiveTo investigate the possible mechanism of modified Gegen Qinlian Decoction （葛根芩连汤） in treatment of ulcerative colitis （UC） from the view of intestinal mucosal epithelial barrier damage and epithelial mesenchymal transition. MethodsSixty male C57BL/6J mice were divided into blank group， model group， western medicine control group， and low-， medium-， and high-dose modified Gegen Qinlian Decoction groups， with 10 mice in each group. Except for the blank group， 3% dextran sodium sulfate （DSS） was used to induce colitis model by free drinking for 7 days， and on the first day of modelling， 6， 12， and 24 g/（kg·d） of modified Gegen Qinlian Decoction were given to the low-， medium-， and high-dose groups respectively， 5-aminosalicylic acid （5-ASA） 100 mg/（kg·d） given by gavage to western medicine control group， and 10 ml/kg distilled water were given to blank and model group by gavage， once a day for 7 days. Body mass of mice was recorded and disease activity index （DAI） scores were performed daily. The mice were anesthetized after 24h of the last administration and the colon was taken to observe the length of colon， HE staining was applied to observe the damage of colonic mucosa and score pathological states， Masson staining to detect the deposition of colonic collagen fibers， immunofluorescence to observe the distribution of F-actin in colonic mucosal epithelium， and immunohistochemistry to detect the expression of tight junction protein ZO-1， Occludin， E-cadherin and Vimentin. ResultsCompared with the blank group at the same time， the percentage of body mass of mice in the model group on day 7 of modelling significantly reduced and the DAI score was significantly increased （P＜0.01）； compared with the model group at the same time， the body mass of mice in the western medicine control group and all of modified Gegen Qinlian Decoction groups decreased， and the DAI scores of mice in the western medicine control group and the high-dose modified Gegen Qinlian Decoction group decreased （P＜0.05 or P＜0.01）； compared with the same time of mice in the low-dos Gegen Qinlian Decoction group， the body mass of mice in the high-dose Gegen Qinlian Decoction group and the western medicine control group significantly elevated （P＜0.05）. Compared with the blank group， the length of the colon of mice in the model group was significantly shortened， the pathological score and the percentage of collagen area were significantly increased， the average fluorescence intensity of F-actin was reduced， the protein levels of ZO-1， Occludin and E-cadherin in the colon tissue decreased， and the protein level of Vimentin elevated （P＜0.01）. Compared with the model group， the length of colon significantly increased， patholo-gical score， collagen area percentage decreased， ZO-1， Occludin， E-cadherin protein levels increased and Vimentin levels decreased in all medicated groups； the average fluorescence intensity of F-actin increased in the western medicine control group and the middle- and high-dose Gegen Qinlian Decoction groups （P＜0.05 or P＜0.01）. Compared with the low-dose Gegen Qinlian Decoction group， the proportion of collagen fibre area in the middle-dose Gegen Qinlian Decoction group and the western medicine control group reduced； the mean fluorescence intensity of F-actin increased in the middle-dose Gegen Qinlian Decoction group； the protein levels of ZO-1 and E-cadherin increased in the western medicine control group， and the protein levels of ZO-1 increased in the high-dose Gegen Qinlian Decoction group （P＜0.05）. Compared with the medium-dose Gegen Qinlian Decoction group， the protein levels of ZO-1 elevated in the high-dose Gegen Qinlian Decoction group （P＜0.05）. Comapred with the high-dose Gegen Qinlian Decoction group， level of E-cadherin and Vimentin protein of the western medicine control group increased （P＜0.05）. ConclusionModified Gegen Qinlian Decoction was able to reduce colonic inflammation and mucosal barrier damage and inhibit the process of epithelial mesenchymal transition in mice models of ulcerative colitis， which may be one of its action mechanisms .