Objective To draw the genome-wide distribution and remodeling characteristics of H3K27me3 silencers in signet-ring cell carcinoma of the stomach(SRCC)through epigenetic sequencing technology,and to investigate their roles in transcriptional regulation in order to elucidate the regulatory mechanism of SRCC malignant progression.Methods The study was conducted on 35 gastric samples obtained by gastroendoscopic biopsy(15 normal and 20 SRCC tissues)from Department of Gastroenterology of Army Medical Center of PLA between January 2021 and December 2023.Multi-omics analyses,including assay for transposase-accessible chromatin with high-throughput sequencing(ATAC-seq),cleavage under targets and tagmentation(CUT&Tag)and transcriptome sequencing(RNA-seq),were performed to identify chromatin accessibility,H3K27me3 silencer regions,and transcriptional changes,with aid of Illumina NovaSeq 6000.H3K27me3 related differentially expressed genes(|Log2FC|>1,FDR<0.05)were screened using DESeq2.Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were employed to analyze the enrichment function,and Homer was employed to identify transcription factor motifs.A regulatory network was constructed using Cytoscape,and then validated using immunohistochemistry to explore its regulatory mechanism.Results H3K27me3 silencers were primarily located in distal intergenic regions(37.06%)in SRCC.Compared with the normal tissues,SRCC showed a significant reduction in H3K27me3 silencer signals(95%CI:1.34～2.30,P=0.007)with 6 257 lost sites(FDR<0.01).Integrating CUT&Tag and RNA-seq revealed 380 up-regulated immune-related genes,particularly in T cell receptor signaling(OR=4.2,95%CI:2.8～6.3,P=0.002).Immunohistochemistry confirmed elevated expression of transcription factor EHF(P<0.05).Conclusion There is the remodeling of H3K27me3 silencers in SRCC,and EHF may potentially play a crucial role in the SRCC malignant progression.

Objective To identify the enhancer profile marked by histone H3K27ac modification in high-grade intraepithelial neoplasia(HGIN)in order to reveal the novel regulatory mechanism of HGIN pathogensis.Methods Gastric tissue samples were collected from Department of Gastroenterology of Army Medical Center of PLA between June 2022 and June 2023,including 14 normal gastric tissues(Nor group),31 HGIN tissues(HGIN group)and 17 gastric cancer tissues(GC group).Cleavage under targets and tagmentation(CUT&Tag)technique was employed to capture enhancer regions modified by histone H3K27ac.Multi-omics analysis was performed to identify HGIN-specific active enhancers and their potentially regulated genes.Immunohistochemical profiling was performed to assess differential expression of the gene of interest across clinically stratified specimens,combined with CRISPR-dCas9-mediated ablation of active enhancers to monitor the gene of interest transcriptional dynamics and validate enhancer-mediated regulatory mechanisms.Results Epigenomic sequencing obtained the data with excellent quality,and indicated that obvious remodeling was observed in H3K27ac enhancers in HGIN and GC groups(P<0.05),though no significant difference in the genome-wide distribution of H3K27ac modification among the 3 groups.Combining transcriptome data revealed that enhancer remodeling may up-regulate the expression of the proliferation-related target gene,CD24,in the HGIN tissue;while,inhibiting enhancer activity can notably reduce CD24 expression level(P<0.05).Immunohistochemical assay displayed a positive correlation between the expression levels of CD24 and Ki-67(P<0.001).Conclusion The remodeling of H3K27ac enhancer represents a significant epigenetic feature of the transformation from normal condition to HGIN.Remodeling of H3K27ac enhancer up-regulates CD24,which may facilitate the abnormal proliferation of gastric epithelial cells.

Objective To analyze clinical characteristics of mesenchymal-subtype gastric cancer(Mes-GC)by integrating multi-omics data and explore the characteristics of tumor cells and microenvironment associated with the risk for recurrence.Methods Gastric tumor tissue samples were collected from the patients who visited Department of Gastroenterology of Army Medical Center of PLA from January 2022 to December 2023.Transcriptome and genome sequencing were applied for these tissue samples,including 19 cases of diffuse-type gastric cancer,22 cases of intestinal-type gastric cancer,and 23 cases of mixed-type gastric cancer patients.Bioinformatics analysis was employed to investigate the differences in clinical characteristics and tumor microenvironment between Mes-GC and non-mesenchymal-subtype gastric cancer(non-Mes-GC)by integrating data resources including The Cancer Genome Atlas(TCGA),Gene Expression Omnibus(GEO),and National Genomics Data Center(NGDC).Results Compared to non-Mes-GC patients,Mes-GC ones were characterized by later clinical stages,deeper tumor infiltration,and higher rates of lymph node metastasis.Kaplan-Meier survival analysis confirmed that Mes-GC patients were associated with shorter survival time,poor prognosis as well as increased risk of cancer recurrence(P<0.05).Single-cell RNA sequencing data revealed that tumor cells in Mes-GC showed higher expression levels of the genes related to stemness,metastasis(P<0.05),and epithelial-mesenchymal transition(EMT).And in the tumor microenvironment,there were significant more myeloid cells,smooth muscle cells,endothelial cells and fibroblasts,with the most pronounced elevation in the proportion of fibroblasts(P<0.05).Moreover,the patients with larger proportion of fibroblasts were associated with poorer prognosis.Conclusion Mes-GC tumor cells exhibit higher stemness and EMT characteristics,and stromal cells such as myeloid cells,endothelial cells,and fibroblasts are enriched in the tumor microenvironment.These features may be key factors contributing to poor prognosis and high recurrence rate of Mes-GC.

Objective To identify the enhancer landscape marked by histone H3K27ac modifications in diffuse-type gastric cancer(DGC)tissues,and to elucidate the epigenetic remodeling mechanisms by which active enhancers regulate cachexia-related genes.Methods Gastric mucosal tissue samples were collected from Department of Gastroenterology of Army Medical Center of PLA during January 2022 to March 2023,including 10 normal gastric mucosa tissues(Normal group),10 DGC tissues diagnosed with cachexia(DGC group),and 10 organoids derived from DGC tissues(Organoid group).Using H3K27ac chromatin targeting cleavage and tagmentation(CUT&Tag)technology,genomic modification regions were captured to screen specific active enhancers and their potential target genes in DGC tissues.CRISPR-dCas9 gene editing technology was used to intervene with the enhancers,and the expression of target genes was detected with Western blotting and qRT-PCR.Sixteen female SPF-grade BALB/c Nude mice(6～8 weeks old,weighing 18～21 g)were utilized to establish an orthotopic xenograft tumor model using the human diffuse-type gastric cancer cell line MKN45.Cachexia-related phenotypes were evaluated in 3 groups:normal group(n=4),silencing group(n=6),and control group(n=6).Results Significant differential enhancer regions were identified between DGC and normal gastric mucosa tissues.DGC tissues exhibited a marked increase in enhancer abundance(P<0.05)and signal intensity when compared with the normal counterparts.Integrated analysis of transcriptome data revealed that some of these active enhancers up-regulated the expression of GDF15,a cachexia-associated target gene in DGC.Targeted silencing of the active enhancer of GDF15 using CRISPR/dCas9-KRAB plasmid technology resulted in a significant reduction in GDF15 expression at both mRNA levels(P<0.05)and protein.Results from orthotopic transplantation experiments of DGC demonstrated that silencing of active enhancers alleviated the cachexia phenotype in nude mice(P<0.05).Conclusion DGC exhibits enhancer remodeling,which regulates the expression of the cachexia-associated gene GDF15,and thereby contributes to the pathogenesis and progression of cancer cachexia.

Purpose To study the safety and clinical value of adenosine stress myocardial perfusion imaging(MPI)in evaluating myocardial ischemia in children with Kawasaki disease.Materials and Methods A total of 78 children with a history of Kawasaki disease and coronary artery aneurysm confirmed by echocardiography and coronary angiography were prospectively studied in Children's Hospital of Fudan University from August 2019 to February 2021.Adenosine stress MPIs were performed to analyze the safety of adenosine stress test and its sensitivity and specificity in detecting significant coronary artery stenosis(≥75%),and the positive rate of adenosine stress MPIs in different groups of coronary artery stenosis were further compared.Results All 78 children completed adenosine stress test without serious side effects.Among 78 children with adenosine stress imaging,44 patients with negative adenosine stress imaging did not undergo rest imaging,which reduced radiation exposure by 56.4%(44/78).The sensitivity and specificity of adenosine stress MPIs in detecting significant coronary artery stenosis were 66.7%and 60.6%(40/66),respectively.Adenosine stress MPIs were positive in 21 cases(21/52,40.3%)in non-stenosis group,5 cases(35.7%)in mild to moderate stenosis group,and 8 cases(66.7%)in significant stenosis group.There was no significant difference in the positive rate among the three groups(χ2=3.169,P=0.205).Conclusion Adenosine stress test is safe and feasible in children.The stress-first imaging strategy can reduce radiation exposure.Adenosine stress MPI has important clinical value in evaluating and monitoring myocardial ischemia in children with Kawasaki disease complicated with coronary aneurysm.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons in the brain and spinal cord. One important aspect of ALS pathogenesis is superoxide dismutase 1 (SOD1) mutant-mediated mitochondrial toxicity, leading to apoptosis in neurons. This study aimed to evaluate the neural protective synergistic effects of ginsenosides Rg1 (G-Rg1) and conditioned medium (CM) on a mutational SOD1 cell model, and to explore the underlying mechanisms. We found that the contents of nerve growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor significantly increased in CM after human umbilical cord mesenchymal stem cells (hUCMSCs) were exposed to neuron differentiation reagents for seven days. CM or G-Rg1 decreased the apoptotic rate of SOD1^G93A-NSC34 cells to a certain extent, but their combination brought about the least apoptosis, compared with CM or G-Rg1 alone. Further research showed that the anti-apoptotic protein Bcl-2 was upregulated in all the treatment groups. Proteins associated with mitochondrial apoptotic pathways, such as Bax, caspase 9 (Cas-9), and cytochrome c (Cyt c), were downregulated. Furthermore, CM or G-Rg1 also inhibited the activation of the nuclear factor-kappa B (NF-κB) signaling pathway by reducing the phosphorylation of p65 and IκBα. CM/G-Rg1 or their combination also reduced the apoptotic rate induced by betulinic acid (BetA), an agonist of the NF-κB signaling pathway. In summary, the combination of CM and G-Rg1 effectively reduced the apoptosis of SOD1^G93A-NSC34 cells through suppressing the NF-κB/Bcl-2 signaling pathway (Fig. 1 is a graphical representation of the abstract).
Humans ; NF-kappa B/metabolism* ; Ginsenosides/pharmacology* ; Amyotrophic Lateral Sclerosis/genetics* ; Culture Media, Conditioned/pharmacology* ; Superoxide Dismutase-1 ; Neurodegenerative Diseases ; Neurons/metabolism* ; Apoptosis

The epitranscriptomic mark N⁶-methyladenosine (m⁶A), which is the predominant internal modification in RNA, is important for plant responses to diverse stresses. Multiple environmental stresses caused by the tea-withering process can greatly influence the accumulation of specialized metabolites and the formation of tea flavor. However, the effects of the m⁶A-mediated regulatory mechanism on flavor-related metabolic pathways in tea leaves remain relatively uncharacterized. We performed an integrated RNA methylome and transcriptome analysis to explore the m⁶A-mediated regulatory mechanism and its effects on flavonoid and terpenoid metabolism in tea (Camellia sinensis) leaves under solar-withering conditions. Dynamic changes in global m⁶A level in tea leaves were mainly controlled by two m⁶A erasers (CsALKBH4A and CsALKBH4B) during solar-withering treatments. Differentially methylated peak-associated genes following solar-withering treatments with different shading rates were assigned to terpenoid biosynthesis and spliceosome pathways. Further analyses indicated that CsALKBH4-driven RNA demethylation can directly affect the accumulation of volatile terpenoids by mediating the stability and abundance of terpenoid biosynthesis-related transcripts and also indirectly influence the flavonoid, catechin, and theaflavin contents by triggering alternative splicing-mediated regulation. Our findings revealed a novel layer of epitranscriptomic gene regulation in tea flavor-related metabolic pathways and established a link between the m⁶A-mediated regulatory mechanism and the formation of tea flavor under solar-withering conditions.
RNA/metabolism* ; Epigenome ; Plant Proteins/metabolism* ; Plant Leaves/metabolism* ; Camellia sinensis/metabolism* ; Flavonoids ; Terpenes/metabolism* ; Tea/metabolism* ; Gene Expression Regulation, Plant

Oolong tea is a semi-fermented tea with strong flavor, which is widely favored by consumers because of its floral and fruity aroma as well as fresh and mellow taste. During the processing of oolong tea, withering is the first indispensable process for improving flavor formation. However, the molecular mechanism that affects the flavor formation of oolong tea during withering remains unclear. Transcriptome sequencing was used to analyze the difference among the fresh leaves, indoor-withered leaves and solar-withered leaves of oolong tea. A total of 10 793 differentially expressed genes were identified from the three samples. KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in flavonoid synthesis, terpenoid synthesis, plant hormone signal transduction and spliceosome pathways. Subsequently, twelve differentially expressed genes and four differential splicing genes were identified from the four enrichment pathways for fluorescence quantitative PCR analysis. The results showed that the expression patterns of the selected genes during withering were consistent with the results in the transcriptome datasets. Further analysis revealed that the transcriptional inhibition of flavonoid biosynthesis-related genes, the transcriptional enhancement of terpenoid biosynthesis-related genes, as well as the jasmonic acid signal transduction and the alternative splicing mechanism jointly contributed to the flavor formation of high floral and fruity aroma and low bitterness in solar-withered leaves. The results may facilitate better understanding the molecular mechanisms of solar-withering treatment in flavor formation of oolong tea.
Camellia sinensis/genetics* ; Gene Expression Profiling ; Plant Leaves ; Plant Proteins/metabolism* ; Taste ; Tea ; Transcriptome/genetics*

Objective:To explore the application situation and hotspots of the Q method in the field of nursing research and provide a reference for the nursing workers in China to learn and apply the Q method.Methods:Literature retrieval was conducted based on Web of Science, and statistical analysis was conducted on the number of articles published, countries, journals, institutions, authors, highly cited papers, research directions and high-frequency co-occurrence words by using the analysis function of Web of Science and visual imaging of VOSviewer.Results:A total of 686 authors from 25 countries and 23 institutions published 326 papers in 157 journals applying the Q method in nursing research, showing an overall increasing trend. South Korea (12.27%) , the United States (7.98%) , the United Kingdom (7.06%) ranked the highest number of articles published, and China ranked the sixth (3.68%) The number of citations per article was the highest in the United Kingdom (33.78 times) , followed by Canada (19.67 times) . Psychology and health care were the most interdisciplinary subjects. The research hotspots expanded from methodological discussions to nursing education, clinical practice, psychological nursing and health promotion.Conclusions:Q method is still in its infancy in the field of nursing in China and has not been widely promoted and practiced. China's publication volume and influence are relatively weak. In the future, international exchanges and cooperation should be expanded and academic frontiers and research hotspots should be grasped to improve the level and quality of research.

Objective:To explore the potential of Taenia solium (Ts) 14-3-3.3 protein as a candidate molecule for cysticercosis vaccine. Methods:Sixty Kunming mice with the body weight of 18 - 22 g were selected and divided into 3 groups according to their body weight via the random number table method, including normal saline control group (control group), Ts14-3-3.3 recombinant protein vaccine group (vaccine group), and Ts14-3-3.3 recombinant protein vaccine + adjuvant group (vaccine + adjuvant group), with 20 mice in each group. The multi-point subcutaneous injection method was adopted. After the first immunization at 0 week, the booster immunization was carried out twice, a total of 3 times, with an interval of 2 weeks. Four mice in the three groups were killed at 0, 2, 4, 6 and 8 weeks after the first immunization, and the blood of eyeballs and spleen were collected aseptically for serum separation and preparation of spleen lymphocytes suspension [treatment: cell suspension, antigen-stimulate and concanavalin (Con) A-stimulate], respectively. The levels of mouse serum specific immunoglobulin (Ig) G, IgG2a, IgG1 and IgE were detected by indirect enzyme-linked immunosorbent assay (ELISA). The proliferation level of mouse spleen lymphocytes was detected via the CCK-8 method. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-13 and IL-10 in culture supernatant of mouse spleen lymphocytes were determined by double-antibody sandwich ELISA.Results:The IgG, IgG2a, and IgG1 levels of the vaccine and vaccine + adjuvant groups immunized for 2 to 8 weeks were higher than those of the control group, and the above indicators of the vaccine + adjuvant group were higher than those of the vaccine group ( P < 0.05). With the same treatment between the groups, the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13, and IL-10 in the culture supernatant after 2 - 8 weeks of immunization were statistically significantly different ( P < 0.05); the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13 and IL-10 in the culture supernatant of the vaccine and vaccine + adjuvant groups immunized for 2 to 8 weeks were higher than those of the control group, and the above indicators of the vaccine + adjuvant group were higher than those of the vaccine group ( P < 0.05). When treatment was different in the group, the proliferation levels of spleen lymphocytes and the levels of TNF-α, IL-12, IL-13 and IL-10 in the culture supernatant of the antigen-stimulate and ConA-stimulate were higher than those of the cell suspension, and the above indicators of the ConA-stimulate were higher than those of the antigen-stimulate ( P < 0.05). Conclusion:The recombinant protein vaccine of Ts14-3-3.3 can induce an effective immune response in mice.