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 investigate the changes in the tumor microenvironment of pancreatic cancer(PDAC)complicated with diabetes mellitus(DM)in a mouse model of hyperglycemia and orthotopic pancreatic cancer by analyzing transcriptome and single-cell transcriptome data in order to identify potential therapeutic targets.Method By integrating single-cell transcriptome and bulk transcriptome data,bioinformatics analysis was conducted to compare the characteristics of tumor cells and tumor immune microenvironment between PDAC patients with DM(DM group)and those without DM(non-DM group).Twenty male C57BL/6 mice(6 weeks old,weighing 18～20 g)were randomly divided into a hyperglycemic group[STZ group,continuous intraperitoneal injection of 50 mg/kg streptozocin(STZ)(final concentration of 1％)dissolved in citrate buffer],and a control group(Control group,an equivalent volume of citrate buffer without STZ at the same time points),with 10 mice in each group.Tail-tip blood glucose level was measured to monitor glycemic status.After orthotopic inoculation of pancreatic cancer cells in both Control and STZ groups,tumor-infiltrating immune cells were harvested.Flow cytometry was employed to determine the effects of hyperglycemia on:total CD8+T cell and Treg cell populations;CD8+T cell subsets expressing Ki67,TNF-α,granzyme B(GZMB)and IFN-γ;surface expression of PD-1,lymphocyte activation gene-3(LAG-3)and T cell immunoglobulin and mucin domain-3(Tim-3)on CD8+T cells;programmed death-ligand 1(PD-L1)expression on tumor cells;and tumor-associated macrophage surface expression of major histocompatibility complex classⅠ(MHC-Ⅰ)and cluster of differentiation 206(CD206).Results Bioinformatics analysis revealed that,compared to the non-DM group,the genes significantly up-regulated in the DM group were associated with poor prognosis(P＜0.001).The proportion of type 2 ductal cells was increased in the DM group,exhibiting higher levels of copy number variation(P＜0.001).In the tumor immune microenvironment of the DM group,there was an increase in the proportion of Treg cells(P＜0.05)and an elevated exhaustion score for CD8+T cells(P＜0.001),accompanied by down-regulated expression of effector molecules,up-regulated expression of inhibitory checkpoints,and a significant increase in the M2 score of M2-like macrophages(P＜0.001).Animal experiments and flow cytometry found that,compared to the Control group,the STZ group had a shorter survival time(P＜0.001),with decreased proportions of total CD8+T cells(P＜0.01)and CD8+T cells expressing Ki67,TNF-α,GZMB and IFN-γ(P＜0.01),increased proportion of Treg cells(P＜0.001),up-regulated expression of PD-1,LAG-3 and Tim-3 on the surface of CD8+T cells(P＜0.001),and up-regulation of PD-L1 on tumor cell surface(P＜0.001)and enhanced expression of CD206 on the surface of tumor-associated macrophages,while down-regulated expression of MHC-Ⅰ(P＜0.001).Conclusion High glucose promotes the formation of an immunosuppressive microenvironment in PDAC,and targeting type 2 ductal cells and immunosuppressive cells in the tumor microenvironment,combined with dual immune checkpoint antibody therapy,may improve patient prognosis.

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.

Objective    To investigate the effect of simvastatin and mechanical pretreatment on intimal hyperplasia of venous graft and its mechanism. Methods    Twelve New Zealand rabbits were selected and randomly divided into 4 groups: a blank control group, a simvastatin topical treatment group, a mechanical precondition group and a combined group (n=3 in each group). Ultrasound was used to evaluate the changes of graft wall and blood flow velocity in the graft, and pathological section was used to evaluate the intimal hyperplasia. Human umbilical cord endodermal cells were cultured in vitro. A simvastatin group and a solvent control group were set to detect YAP phosphorylation, downstream target gene expression and cell proliferation. Results    Vascular ultrasound showed that except the simvastatin topical treatment group, the flow velocity in vein grafts in the other three groups significantly increased 21 days after surgery compared with 7 days after surgery (P<0.01). Pathological sections showed that the thickness of new intima in the simvastatin topical treatment group, mechanical precondition group, combined group and blank control group were 45.56±4.11 μm, 201.28±16.71 μm, 143.57±7.82 μm, 249.45±13.33 μm, respectively, and there were statistical differences compared with the blank control group (P<0.05). In vitro results showed that compared with the solvent control group, cell death was observed in high concentration simvastatin (5 mmol/L) group, cell proliferation was inhibited in low concentration simvastatin (2.5 mmol/L) group (P<0.05), the expression of YAP protein in the simvastatin group was unchanged, but the expression of phosphorylated YAP protein significantly increased (P<0.05), and the expression of downstream target gene ccn1 was down-regulated (P<0.001). Conclusion    Intravascular local application of simvastatin and mechanical preconditioning alone or in combination can inhibit intimal hyperplasia of venous graft. High concentration of simvastatin has cytotoxicity, while low concentration of simvastatin has inhibitory effect on cell proliferation. Simvastatin can inhibit the formation of new intima by inhibiting the entry of YAP into the nucleus and reducing the transcription of cell proliferation-related target gene ccn1.

Objective:To explore the trans-membrane signaling mechanism of interleukin-6 (IL-6)-induced osteogenic differentiation and calcification of human umbilical artery smooth muscle cells (HUASMCs).Methods:HUASMCs were primarily cultured in vitro and were stimulated with IL-6, IL-6+solutable IL-6 receptor (sIL-6R), IL-6+sIL-6R+solutable gp130 (sgp130), or vehicle (blank control). Alizarin red and Von Kossa staining were used for detecting cell calcification, Western blot was used to test the protein expression of tissue-nonspecific alkaline phosphatase (TNAP), osteopontin (OPN), bone morphogenetic protein-2 (BMP-2) and Runt related transcription factor 2 (Runx2), and immunofluorescence was used to examine the mIL-6R expression of HUASMCs. The comparison of measurement date between the two groups was conducted by t-test. The comparison of measurement date between multiple groups was conducted by one-way analysis of variance (ANOVA). Results:The intensity severity of calcification stain was IL-6+sIL-6R group >IL-6+sIL-6R+sgp130 group>IL-6 group=blank control. After stimulated for 12 hours, the TNAP expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.44±0.08), (0.52±0.14), (0.84±0.16) and (0.55±0.10) respectively ( F=290.96, P<0.001). After stimulated for 3 days, the OPN expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.61±0.84), (0.95±0.16), (1.65±0.24) and (0.99±0.10) respectively ( F=507.72, P<0.001). After stimulated for 12 hours, the BMP-2 expression in blank control, IL-6 group, IL-6+sIL-6R group, IL-6+sIL-6R+sgp130 group were (0.77±0.05), (1.69±0.16), (2.81±0.26) and (0.57±0.12) respectively ( F=959.09, P<0.001). After stimulated for 3 days, the Runx2 expression in blank control, IL-6 group, IL-6+sIL-6R group,IL-6+sIL-6R+sgp130 group were (0.57±0.03) , (0.92±0.10), (1.31±0.13) and (0.66±0.06) respectively ( F=1141.27, P<0.001). Comparing with Jurkat cells (positive control) and CEM cells (negative control), HUASMCs limited expressed mIL-6R. Conclusion:IL-6 may induce HUASMCs osteogenic differentiation and calcification mainly via the sIL-6R-mediated trans-signaling pathway.

Objective:To analyze the effect of Croton leaf on ERK1/2 pathway in hippocampal of rats with cerebral ischemia-reperfusion.Methods:A total of 216 SD male rats were divided into sham operation group, model group, nimodipine group, low-dose, medium-dose and high-dose groups of croton leaf according to random nubmer table method, with 36 rats in each group. The MACO model of rats was prepared by the method of wire embolization. The high, medium and low dose groups were intragastrated with the water decoction 0.06 g/ml, 0.12 g/ml and 0.18 g/ml of Croton leaf; nimodipine group was intragastrated with nimodipine suspension 1.08 g/L; sham operation group and model group were intragastrated with equal volume of normal saline. Garcia JH score was used to conduct neurological function score, and HE staining was used to observe the morphology of hippocampal neurons after 7 days of continuous administration. Apoptosis of hippocampal CA3/DG region was detected by TUNEL assay. Western Blot was used to detect the expression of ERK1/2 and p-ERK1/2 proteins.Results:Compared with the model group at simultaneous point, the neurological function scores of low-dose, medium-dose and high-dose groups of Croton leaf increased ( P<0.01), the number of apoptotic cells decreased ( P<0.01), the expression of p-ERK1/2 / ERK1/2 [1 d: (0.22±0.03, 0.34±0.02, 0.46±0.01 vs. 0.19±0.02); 3 d: (0.38±0.02, 0.50±0.02, 0.68±0.02 vs. 0.27±0.02); 7 d: (0.29±0.03, 0.43±0.02, 0.59±0.02 vs. 0.21±0.03)] in hippocampus of the low-dose, medium-dose and high-dose groups of Croton leaf significantly increased ( P<0.01). Conclusion:Croton leaf could regulate the expression of ERK1/2 pathway protein upward, effectively improve the neural function and resist the apoptosis of hippocampal CA3/DG area of rats with cerebral ischemia-reperfusion injury.

Objective:To understand the symptom perception and coping experience of postoperative delirium in patients with Stanford type A aortic dissection (TAAD) .Methods:Using the purposive sampling method, a total of 13 patients with delirium after Stanford type A aortic dissection who were admitted to Department of Cardiovascular Surgery of Henan Provincial Chest Hospital from July 2020 to July 2021 were selected as the research objects. Semi-structured interviews were conducted with patients using the phenomenological research method and the interview data were analyzed using the Colaizzi 7-step analysis method.Results:Postoperative delirium in patients with TAAD was classified into four sub-themes, such as confusion, physical and mental discomfort, cognitive disturbance and death perception. The coping experience was classified into three sub-themes, such as communication barriers, lack of encouragement and support and need for safe companionship.Conclusions:Patient with delirium after TAAD have many uncomfortable symptoms and long for support and help.

Membrane-disruptive peptides/peptidomimetics (MDPs) are antimicrobials or anticarcinogens that present a general killing mechanism through the physical disruption of cell membranes, in contrast to conventional chemotherapeutic drugs, which act on precise targets such as DNA or specific enzymes. Owing to their rapid action, broad-spectrum activity, and mechanisms of action that potentially hinder the development of resistance, MDPs have been increasingly considered as future therapeutics in the drug-resistant era. Recently, growing experimental evidence has demonstrated that MDPs can also be utilized as adjuvants to enhance the therapeutic effects of other agents. In this review, we evaluate the literature around the broad-spectrum antimicrobial properties and anticancer activity of MDPs, and summarize the current development and mechanisms of MDPs alone or in combination with other agents. Notably, this review highlights recent advances in the design of various MDP-based drug delivery systems that can improve the therapeutic effect of MDPs, minimize side effects, and promote the co-delivery of multiple chemotherapeutics, for more efficient antimicrobial and anticancer therapy.

【Objective】 To explore the clinical effectiveness of platelet transfusion during pediatric extracorporeal membrane oxygenation (ECMO). 【Methods】 Neonates and children treated with ECMO and received platelets transfusion at least once in the Department of Cardiothoracic Surgery, Shanghai Children′s Medical Center in 2020 were enrolled in our research. Platelet count was measured 24 hours before and after each platelet transfusion, and the corrected count increment (CCI) was calculated for effectiveness estimation of platelet transfusion. The research objects were divided into three groups based on the platelet count before transfusion, and the effective rates of each group were calculated. 【Results】 Thirty-seven patients received 79 platelet transfusions (2.14±1.21 on average), among which 39 were effective (49.37%) and 40 were refractory (50.63%). The effective rates of group 1, group 2 and group 3 were 1, 67.74%, 40.91%, and 34.62%, respectively. 【Conclusion】 The restricted platelet transfusion strategy, on the premise of ensuring life safety, is preferred for children treated with ECMO.