Objective: To evaluate the in vitro procoagulant efficacy and in vivo potential thrombogenic risk of self-prepared activated prothrombin complex concentrate (aPCC). Methods: The preparation was administered at a dose of 0.5 IU per milliliter of plasma. For in vitro assays, the self-prepared aPCC concentrate was added to plasma spiked with factorⅧ (FⅧ) inhibitors. Calibrated automated thrombin generation (CAT), prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (Fib) quantification were performed. Normal saline and FEIBA were set as controls to evaluate the procoagulant capacity of the self-prepared aPCC concentrate. For in vivo animal studies, the self-prepared aPCC concentrate was injected into rats via the jugular vein. A ferric chloride (FeCl3) filter paper patch was applied to induce carotid arterial thrombosis. Laser speckle blood flow imaging was used to continuously monitor thrombus formation in the rat carotid artery. Plasma samples were collected from rats for CAT, PT, APTT, TT and Fib testing, with normal saline and FEIBA as controls to evaluate the potential thrombogenic risk of the self-prepared aPCC concentrate. Results: Supplementation with aPCC significantly shortened APTT and PT values in inhibitor-spiked plasma (all P<0.001). There were no statistically significant differences in APTT and PT between FEIBA and self-prepared aPCC (P>0.05). CAT results demonstrated that adding aPCC to inhibitor-containing plasma markedly increased endoge-nous thrombin potential (ETP) and peak thrombin concentration (Peak) (P<0.001), with both parameters higher than those of normal plasma (P<0.05). No significant intergroup difference in ETP was observed between self-prepared aPCC and FEIBA (P>0.05). Carotid blood flow imaging in rats revealed that, compared with FEIBA , thrombi induced by self-prepared aPCC were smaller and less stable, and all underwent spontaneous lysis, accompanied by better restoration of carotid blood perfusion. Conclusion: The self-prepared aPCC restores coagulation function in inhibitor-spiked plasma to a comparable extent as FEIBA , while exhibiting a lower thrombogenic risk.

BACKGROUND:Umbilical cord mesenchymal stem cells present immunomodulatory function and multidirectional differentiation potential.Umbilical cord mesenchymal stem cells can actively improve the chronic low-grade inflammation state in type 2 diabetes mellitus and which is important for preventing the development of type 2 diabetes mellitus progression.OBJECTIVE:To review the research progress of umbilical cord mesenchymal stem cells in the treatment of type 2 diabetes mellitus through immunomodulatory effect.METHODS:Using"umbilical cord mesenchymal stem cells,UC-MSCs,mesenchymal stem cells,MSCs,type 2 diabetes mellitus,T2DM,immunity,insulin resistance,inflammation"as Chinese and English search terms,articles were searched on CNKI,Web of Science and PubMed databases.Relevant reviews,research papers and theses were screened and 88 papers were collected for summarization based on the inclusion and exclusion criteria.RESULTS AND CONCLUSION:Umbilical cord mesenchymal stem cells can effectively delay the occurrence and development of type 2 diabetes mellitus by reducing inflammatory response,inducing macrophages to M2 polarization,increasing the sensitivity of target organs to insulin,improving insulin resistance,and alleviating the dysfunction of islet beta cells through immune regulation.However,before umbilical cord mesenchymal stem cell transplantation can become a routine treatment,large-scale basic and applied basic research is needed to determine the optimal treatment regimen and efficacy.

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.

Systemic mastocytosis (SM) with an associated myelodysplastic/myeloproliferative neoplasm (MDS/MPN) is a rare subtype of myeloid neoplasms. Avapritinib, a potent and selective inhibitor of KIT D816V, is approved for treating advanced systemic mastocytosis (AdvSM). We report a case of a patient with SM and an associated MDS/MPN treated with avapritinib. The patient achieved sustained complete remission (CR) of SM, with persistent molecular negativity for the KIT D816V mutation, but ultimately succumbed to disease progression to chronic myelomonocytic leukemia (CMML). Although avapritinib, a novel targeted therapy, has significantly improved outcomes for SM, the efficacy of treatment for the associated hematologic neoplasm in patients with SM-AHN may be the primary determinant of long-term overall survival and progression-free survival. This report includes a review of relevant literature to provide insights into the clinical diagnosis and management of this rare entity.

BACKGROUND:Umbilical cord mesenchymal stem cells present immunomodulatory function and multidirectional differentiation potential.Umbilical cord mesenchymal stem cells can actively improve the chronic low-grade inflammation state in type 2 diabetes mellitus and which is important for preventing the development of type 2 diabetes mellitus progression.OBJECTIVE:To review the research progress of umbilical cord mesenchymal stem cells in the treatment of type 2 diabetes mellitus through immunomodulatory effect.METHODS:Using"umbilical cord mesenchymal stem cells,UC-MSCs,mesenchymal stem cells,MSCs,type 2 diabetes mellitus,T2DM,immunity,insulin resistance,inflammation"as Chinese and English search terms,articles were searched on CNKI,Web of Science and PubMed databases.Relevant reviews,research papers and theses were screened and 88 papers were collected for summarization based on the inclusion and exclusion criteria.RESULTS AND CONCLUSION:Umbilical cord mesenchymal stem cells can effectively delay the occurrence and development of type 2 diabetes mellitus by reducing inflammatory response,inducing macrophages to M2 polarization,increasing the sensitivity of target organs to insulin,improving insulin resistance,and alleviating the dysfunction of islet beta cells through immune regulation.However,before umbilical cord mesenchymal stem cell transplantation can become a routine treatment,large-scale basic and applied basic research is needed to determine the optimal treatment regimen and efficacy.

Systemic mastocytosis (SM) with an associated myelodysplastic/myeloproliferative neoplasm (MDS/MPN) is a rare subtype of myeloid neoplasms. Avapritinib, a potent and selective inhibitor of KIT D816V, is approved for treating advanced systemic mastocytosis (AdvSM). We report a case of a patient with SM and an associated MDS/MPN treated with avapritinib. The patient achieved sustained complete remission (CR) of SM, with persistent molecular negativity for the KIT D816V mutation, but ultimately succumbed to disease progression to chronic myelomonocytic leukemia (CMML). Although avapritinib, a novel targeted therapy, has significantly improved outcomes for SM, the efficacy of treatment for the associated hematologic neoplasm in patients with SM-AHN may be the primary determinant of long-term overall survival and progression-free survival. This report includes a review of relevant literature to provide insights into the clinical diagnosis and management of this rare entity.

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.