Objective: To investigate the role of calcium/calmodulin-dependent protein kinase Ⅱ delta (CaMKⅡδ) in platelet activation mediated by thrombin and thrombin receptor activator peptide (TRAP). Methods: The CaMKⅡδ-specific inhibitor Hesperadin was used to assess its inhibitory effects on platelet activation using multiple detection methods. These included: analyzing the inhibitory effect of different concentrations of Hesperadin on thrombin- and TRAP-induced platelet aggregation using a platelet aggregometer; measuring CD62P expression levels and calcium mobilization via flow cytometry; and determining dense granule release activity using a microplate reader. Results: Hesperadin at concentrations of 40 μM and 80 μM significantly inhibited thrombin- and TRAP-induced platelet aggregation (P<0.05). Within the concentration range of 20–80 μM, it significantly inhibited α-granule release and TRAP-induced calcium ion mobilization (P<0.05); at 80 μM, it markedly suppressed thrombin-induced platelet calcium ion mobilization and significantly inhibited dense granule release (P<0.05). Conclusion: The experimental results indicate that CaMKⅡδ in platelets is activated and plays a regulatory role in the process of platelet activation induced by thrombin and TRAP.

Objective To map the genome-wide distribution profile of histone H3K27me3 modification in diffuse gastric cancer tissues,identify target genes regulated by H3K27me3,and primarily explore the potential mechanism of its modification reprogramming in the occurrence and development of the tumor.Methods Normal gastric mucosal tissues and diffuse gastric cancer tissues were harvested from the patients who underwent examinations or treatments in the departments of gastroenterology and gastrointestinal surgery of our medical center between 2021 and 2023.There were 14 patients in the normal group(6 males and 8 females,average age of 46 years)and 14 patients in the gastric cancer group(8 males and 6 females,average age of 63 years).Cleavage under target and tagmentation(CUT&Tag)technology was employed to capture genomic regions modified by H3K27me3,and analyze the reprogramming characteristics of these modifications.RNA sequencing data,data from high-throughput chromosome conformation capture(Hi-C)technology,and publicly available single-cell data were integrated to investigate the target genes regulated by the reprogramming of H3K27me3 modifications in diffuse gastric cancer cells.Results The quality of the CUT&Tag and RNA sequencing data met the standards required for subsequent analysis.Histone H3K27me3 modifications in normal gastric mucosa and diffuse gastric cancer tissues were primarily distributed in distal intergenic regions and intronic regions.In gastric cancer tissues,compared to normal tissues,there was significant reprogramming of H3K27me3 modifications,characterized by a marked reduction in overall H3K27me3 signal intensity.The loss of 2 912 H3K27me3 signal peaks might lead to the up-regulation of 822 tumor-associated genes.Among them,56 genes displayed the most significant up-regulation(fold change in signal intensity≥2,P<0.05),with notable enrichment in the mammalian target of rapamycin complex 1(mTORC1)signaling pathway.Specifically,the methionine transporter SLC7A5 and the cystine transporter SLC7A11 were found to have the highest expression levels in gastric cancer tissues.Single-cell data revealed that the abnormal overexpression of SLC7A11 in diffuse gastric cancer was primarily observed in tumor epithelial cells.Further validation using public data and immunohistochemical experiments confirmed the elevated expression of SLC7A11 in diffuse gastric cancer,which is associated with poor prognosis in gastric cancer patients.Conclusion The reprogramming of histone H3K27me3 modification is an important epigenetic characteristic in diffuse gastric cancer.Loss of H3K27me3 signal peaks may up-regulate the expression of SLC7A11 in diffuse gastric cancer cells,and thereby promote tumor progression.

Objective To investigate the diagnostic value of endoscopic sign of light blue crest(LBC)capsuling papillary lesion(LCPL)for high-risk intestinal metaplasia(IM).Methods A total of 314 patients(352 biopsy specimens)who underwent endoscopic examination and biopsy in Department of Gastroenterology of Army Medical Center of PLA from January 2021 to June 2023 were recruited,and HE and HID-AB staining(the golden standard of high-risk IM)were apllied to detect the histological types and IM types.The samples were subsequently divided into chronic inflammation group,low-risk IM group,high-risk IM group,well-differentiated intestinal-type gastric cancer group,and poorly-differentiated intestinal-type gastric cancer group.The positive rate of LCPL in each group and its diagnostic efficacy were analyzed based on endoscopic images of the biopsy sites.Logistic regression analysis was used to investigate the relationship between LCPL sign and high-risk IM,as well as the clinical and pathological features associated with LCPL sign.Receiver operating characteristic(ROC)curve was plotted to evaluate the diagnostic efficacy of LCPL for high-risk IM,using indicators such as sensitivity,specificity,Youden index and area under the curve(AUC).Results The positive rate of the LCPL sign in high-risk IM group was 75.70%,significantly higher than that of the other groups(all P<0.001).Logistic regression analysis showed that LCPL sign was significantly correlated with high-risk IM(OR=30.286,95%CI:13.528～67.804,P<0.001).When the sign was employed in diagnosing high-risk IM,the sensitivity was 69.84%,the specificity was 93.75%,the Youden's index was 0.636,and the AUC value was 0.818(95%CI:0.773～0.857).Besides sensitivity,all above parameters of LCPL sign showed significantly better diagnostic efficacy than those of traditional LBC sign,which is used as a sign for diagnosing IM(P<0.001).Moreover,recognition of LCPL sign was not easily affected by age(OR=1.130,95%CI:0.709～1.800,P=0.607),lesion site(Angular incisure:OR=2.360,95%CI:0.732～7.613,P=0.151;Autrum:OR=2.257,95%CI:0.756～6.744,P=0.145),and presence of peptic ulcers(OR=1.085,95%CI:0.208～5.652,P=0.923).Significantly,94.12%of positive and 66.94%of negative LCPL signs could be rapidly recognized within 3 s(OR=4.536,95%CI:1.372～14.997,P=0.013).Conclusion LCPL sign shows high efficacy and potential clinical application value for high-risk IM in gastric mucosa of endoscopic diagnosis.

Head and neck squamous cell carcinoma (HNSCC) is the most common type of heterogeneous malignant tumor. Over 60% of HNSCC patients are at locally advanced stage at the time of diagnosis. Despite the emergence of advanced diagnosis and treatment measures, the prognosis of patients with recurrent or metastatic HNSCC remains poor. The Period (PER) gene family is an important member of the circadian clock genes, with family members PER1, PER2, and PER3 showing differential expression in HNSCC, participating in biological processes such as proliferation, apoptosis, invasion, or metastasis of tumor cells. In most studies, they exhibit anti-cancer effects and are closely related to the tumor microenvironment, immunotherapy, chronotherapy, etc., making them potential biomarkers. A comprehensive analysis of the expression of PER gene family in HNSCC, its biological role in the occurrence and development of tumor and the corresponding molecular biological mechanism is helpful to explore its potential application value in the diagnosis and treatment of HNSCC.

The immune checkpoint blockade (ICB) targeting on PD-1/PD-L1 has shown remarkable promise in treating cancers. However, the low response rate and frequently observed severe side effects limit its broad benefits. It is partially due to less understanding of the biological regulation of PD-L1. Here, we systematically and comprehensively summarized the regulation of PD-L1 from nuclear chromatin reorganization to extracellular presentation. In PD-L1 and PD-L2 highly expressed cancer cells, a new TAD (topologically associating domain) (chr9: 5,400,000-5,600,000) around CD274 and CD273 was discovered, which includes a reported super-enhancer to drive synchronous transcription of PD-L1 and PD-L2. The re-shaped TAD allows transcription factors such as STAT3 and IRF1 recruit to PD-L1 locus in order to guide the expression of PD-L1. After transcription, the PD-L1 is tightly regulated by miRNAs and RNA-binding proteins via the long 3'UTR. At translational level, PD-L1 protein and its membrane presentation are tightly regulated by post-translational modification such as glycosylation and ubiquitination. In addition, PD-L1 can be secreted via exosome to systematically inhibit immune response. Therefore, fully dissecting the regulation of PD-L1/PD-L2 and thoroughly detecting PD-L1/PD-L2 as well as their regulatory networks will bring more insights in ICB and ICB-based combinational therapy.

Objective To evaluate the relationship between annexin 1 (ANXA1) and the endogenous protective mechanism during intestinal epithelial cell injury induced by endotoxiu.Methods The intestinal epithelial cells at the logarithmic growth phase were seeded in culture palates and randomly divided into 4 groups (n =36 each) using a random number table:control group (group C),cell injury group (group I),ANXA1 overexpression group (group OE),and ANXA1 silencing group (group S).Lentivirus with ANXA1 overexpression and silencing was transfected into intestinal epithelial cells to construct a stable cell line.In I,OE and S groups,endotoxin was added with the final concentration of 100 μg/ml,and the cells were then incubated for 24 h to establish the cell injury model.The culture medium was changed,and the cells were then incubated for 24 h in group C.The cell apoptosis was detected by flow cytometry,the cell permeability was determined by Transwell assay,and the cell viability was evaluated by methyl thiazolyl tetrazolium assay.The apoptosis rate was calculated.Results Compared with group C,the apoptosis rate was significantly increased,and the cell permeability and viability were significantly decreased in I,OE and S groups (P＜0.05).Compared with group Ⅰ,the apoptosis rate was significantly decreased,the cell permeability and viability were significantly increased in group OE,and the apoptosis rate was significantly increased,and the cell permeability and viability were significantly decreased in group S (P＜0.05).Conclusion ANXA1 is involved in the endogenous protective mechanism during intestinal epithelial cell injury induced by endotoxin.

Objective To explore the expression of heat shock protein (HSP) 90α in outside of different metastatic hepatocellular carcinoma (HCC) cell lines and its role in the cells migration and invasion.Methods The expression of HSP90α was detected by Western blot analysis in conditioned media of MHCC97L and MHCC97H with low and high metastatic HCC cell lines.A small molecule cell-impermeant HSP90 inhibitor DMAG-N-oxide was used to inhibit extracellular HSP90α.Changes of the cells migratory and invasive capability were assessed by in vitro motility and invasion assay.The endogenous matrix metalloproteinase 2 (MMP-2) was demonstrated by Zymography.The expression of extracellular co-chaperone HSP70 and MMP-2 were tested by Western blots and the association between HSP90α,HSP70 and MMP-2 was analyzed by immunoprecipitation.The effects of HSP70 knockdown by siRNA,with or without MMP-2 inhibitor Batimastat,on the level of active MMP-2 and cell migration and invasion were also evaluated.Results HSP90α can express both inside and outside of different metastatic HCC cell lines,and the level of expression was consistent with metastasis potentials.After MHCC97-H cells were treated with a special HSP90α inhibitor DMAG-N-oxide for 24 h,the average migratory cell numbers (28.11 ±3.56) had a significantly reduction,compared with those without treatment group (80.12±4.16) and empty control group (82.24±4.12),respectively (P ＜ 0.01).In vitro invasion assay showed the average invaded cell numbers in treatment group (36.54±4.12) were more fewer than without treatment group (95.12±3.48) and empty control group (101.1 1±3.36),respectively (P =0.017),and accompanying with decreasing of the extracellular MMP-2 activity.HSP70 and MMP-2 could express outside of MHCC97-H cells and interact with HSP90α.Small molecular interfere RNA (siRNA) dramatically inhibited HSP70 expression and reduced the interaction HSP90α with MMP-2 and MMP-2 activity outside MHCC97-H cells,and also suppressed MHCC97-H cells migration and invasion.In addition,combining MMP-2 inhibitor had additive inhibition effects.Conclusion Extracellular HSP90α and HSP70 form chaperone complex to assist in MMP-2 activation and increases HCC cells migration and invasion,which maybe a novel therapeutic target against metastatic HCC.

Objective To evaluate the relationship between extracellular signal-regulated kinase (ERK)and ketamine-induced apoptosis in rat hippocampal neurons.Methods Sprague-Dawley rats at 18 days of gestation were anesthetized.The fetal rats were obtained under the sterile condition and decapitated.The hippocampal neurons were isolated and primarily cultured for 5 days,and were seeded in 6-well plates (2 ml/well) or in 96-well plates (100μl/well) at a density of 5 × 105/ml.The cells were randomly divided into 4 groups (n =18 each):control group (group C),fibroblast growth factor (FGF-2,an ERK agonist) group (group F),ketamine group (group K) and FGF-2 + ketamine group (group FK).The cells were cultured in the plain culture medium in group C.FGF-2 50 ng/ml was added to the culture medium in group F.Ketamine was added to the culture medium in group K.FGF-2 50 ng/ml was added to the culture medium at 20 min before ketamine 100 μmol/L was added in group FK.The phosphorylation of ERK in hippocampal neurons was detected by Western blot at 10 min after treatment.At 24 h after treatment,the neuronal apoptosis was detected by Hoechst33342/PI staining,and the cell survival rate was detected by MTT assay.The apoptosis rate was calculated.Results Compared with group C,the phosphorylation of ERK in hippocampal neurons and the cell survival rate was significantly decreased and the apoptosis rate was increased in K and FK groups (P ＜ 0.05).There was no significant difference in the parameters mentioned above between F and C groups (P ＞ 0.05).The phosphorylation of ERK in hippocampal neurons and the cell survival rat was significantly higher and the apoptosis rate was lower in group FK than in group K (P ＜0.05).Conclusion Ketamine induces apoptosis in rat hippocampal neurons by inhibiting activation of ERK in hippocampal neurons.