BACKGROUND AND AIM:The benefit of combining linaclotide with polyethylene glycol (PEG) for improving bowel preparation quality and adenoma detection in patients at risk for inadequate bowel preparation remains uncertain. This study aimed to evaluate its efficacy and safety. METHODS:From August 2022 to July 2023, a multicenter, randomized trial assigned participants to a 2-day linaclotide or placebo plus PEG regimen. The primary endpoints included adequate bowel preparation rate and mean number of adenomas detected per colonoscopy. RESULTS:The modified intention-to-treat (mITT) (n=672) and per-protocol (n=574) analyses showed no significant differences in adequate bowel preparation rates (mITT: OR 1.26, 95% CI 0.73-2.16, P=0.406; per-protocol: OR 1.46, 95% CI 0.80-2.66, P=0.222) or the mean number of adenomas (mITT: mean difference -0.02, 95% CI -0.19-0.15, P=0.832; per-protocol: mean difference -0.06, 95% CI -0.25-0.13, P=0.530) between groups. However, linaclotide and PEG increased the mean number of polyps in the right colon (0.54 ± 1.9 vs. 0.27 ± 0.8; mean difference -0.27, 95% CI -0.49- -0.05, P=0.016), particularly in patients aged ≥70 years (mean difference -1.30, 95% CI -2.48- -0.13, P=0.033). CONCLUSIONS:A 2-day linaclotide regimen failed to improve the primary endpoints of bowel preparation adequacy or adenoma detection in high-risk patients.

BACKGROUND:Immune checkpoint inhibitors (ICIs) demonstrate limited efficacy in hepatocellular carcinoma (HCC), largely attributable to a profoundly immunosuppressive tumour microenvironment (TME). OBJECTIVE:To investigate the kinase never-in-mitosis A-related kinase 9 (NEK9) as a potential tumour-intrinsic driver of immune evasion and therapeutic target. DESIGN:NEK9 expression and its clinical relevance were analysed in HCC cohorts. Functional investigations employed genetic and specific pharmacological approaches in HCC cell lines and orthotopic mouse models. The TME was comprehensively profiled using single-cell RNA sequencing, flow cytometry and multiplex immunohistochemistry. Mechanistic insights were gained through co-immunoprecipitation, phosphoproteomic analysis and kinase assays. Synergy between NEK9 inhibition and programmed death-ligand 1 (PD-L1) blockade was quantitatively assessed using zero interaction potency (ZIP) reference models. RESULTS:NEK9 was significantly upregulated in HCC and correlated with poor survival, diminished intratumoral CD8+ T cell infiltration and increased myeloid-derived suppressor cells (MDSCs). Mechanistically, NEK9 directly phosphorylated TRIM28 and USP46, stabilising nuclear factor-κB2 (NF-κB2) and driving PD-L1 and CXCL1 transcription, thereby promoting CD8+ T cell dysfunction and CXCR2-dependent recruitment of MDSCs. Pharmacological NEK9 inhibition destabilised NF-κB2 and reversed the immunosuppressive TME. Importantly, two novel small-molecule NEK9 inhibitors (MIPO, FPTP) were identified, which synergised strongly with anti-PD-L1 therapy, enhancing CD8+ T cell effector function and tumour suppression in vivo. CONCLUSIONS:NEK9 is a druggable driver of immune evasion in HCC. Targeting NEK9 remodels the immunosuppressive TME and synergises with PD-L1 blockade, offering a promising strategy to overcome ICI resistance.

The mitotic index is a critical indicator in grading gastrointestinal stromal tumors (GIST). Conventional microscopy-based mitosis counting is labor-intensive and exhibits interobserver variability, necessitating automation. However, existing models have proven unsuitable for GIST spindle cells. To address this limitation, we developed a machine learning method for automated mitosis detection in GIST. A GIST image database, annotated with 13,965 mitotic cells, was first established. Following nuclei segmentation, feature extraction, and feature selection, six different algorithms were employed to train mitosis detection models on images at both 10 × and 40 × magnification levels. The Radial Basis Function Support Vector Machine (SVM-RBF) achieved the best performance under both magnifications (10 ×: F1 = 0.83; 40 ×: F1 = 0.89). Slide-level mitosis counting was then performed via a two-step cascaded dual-scale approach, in which the 10 × model first identified Regions of Interest (ROIs), followed by precise detection and counting using the 40 × model. Slide-level validation showed a moderate correlation between automated and manual mitosis counts (r = 0.4705) and a strong correlation between the automated counts and Ki-67 expression (r = 0.6187). SHAP interpretability analysis confirmed that the model's decision-making basis closely aligned with pathologists' diagnostic criteria, including nuclear membrane disintegration, chromatin condensation, and chromosomal alignment. In summary, this study establishes the first automated framework for mitotic cell detection and counting in GIST. It underscores the clinical potential of traditional machine learning for targeted pathological applications and demonstrates favorable interpretability.

Small gastric subepithelial tumors (SETs) in the stomach can be managed through surveillance or resection. However, it is still controversial how often the lesion would progress if left untreated. This study aimed to evaluate the progression rate of small SETs and identify risk factors influencing tumor growth. PubMed, Cochrane Library, Web of Science, and Scopus were searched for relevant studies until March 2023. Patient information, endoscopic features of SETs, and surveillance information were extracted from each included study. A random-effects model was applied along with subgroup and sensitivity analyses. Based on 14 studies with 5405 SETs smaller than 3.5 cm, the annual incidence of size increase was 4.0 (95

Genetically modified intestinal organoids are being explored as potential surrogates of immortalized cell lines and gene-engineered animals. However, genetic manipulation of intestinal organoids is time-consuming, and the efficiency is far beyond satisfactory. To ensure the yield of the genetically modified organoids, large quantity of starting materials is required, and the procedure usually takes more than 10 days. Two major obstacles that restrict the genetic delivery efficiency are the three-dimensional culture condition and that the genetic delivery is carried out in cell suspensions. In the present study, we introduce a novel highly efficient strategy for building genetically modified intestinal organoids in which genetic delivery was performed in freshly established monolayer primary intestinal epithelial cells under two-dimensional conditions and subsequentially transformed into three-dimensional organoids. The total procedure can be finished within 10 hr while displaying much higher efficiency than the traditional methods. Furthermore, this strategy allowed for the selection of transgenic cells in monolayer conditions before establishing high-purity genetically modified intestinal organoids.

Background: Vonoprazan, a novel acid suppressant, has recently emerged as a regimen for eradicating Helicobacter pylori. However, uncertainties exist about the effectiveness and safety of VPZ-based regimens compared with those of bismuth-based quadruple therapy in eradicating H. pylori. The present meta-analysis was performed to compare the effectiveness and safety of vonoprazan-based regimens with those of bismuth quadruple therapy in eradicating H. pylori. Materials and methods: All randomized controlled trials and non-randomized controlled trials comparing the vonoprazan-based therapy with the bismuth quadruple therapy were included in this meta-analysis. Information was also extracted by two evaluators, and if heterogeneity existed, a random-effects model was used to calculate the combined relative ratio and 95% confidence interval; otherwise, a fixed-effects model was used. And subgroup analyses were performed to explore the sources of heterogeneity. Results: A total of 10 studies, comprising 2587 patients were included in the meta-analysis. The results showed that the combined eradication rate of patients treated with the vonoprazan-based regimen was significantly higher than that of patients treated with bismuth quadruple therapy, in both intention-to-treat and per-protocol analyses, and the differences were statistically significant. Among the intention-to-treat analyses results: (90.28% vs. 83.64% [odds ratio (OR) = 1.85, 95% confidence interval (CI) (1.27, 2.70), p = 0.001]); in the per-protocol analyses: (94.80% vs. 89.88%, [OR = 2.25, 95% CI (1.37, 3.69), p = 0.001]). The occurrence of adverse events was significantly lower in patients treated with vonoprazan-based regimens than in those treated with bismuth quadruple therapy, (14.50% vs. 25.89%, [OR = 0.49, 95% CI (0.32, 0.75), p = 0.001]). Conclusions: For eradicating H. pylori, vonoprazan-based regimens are remarkably advantageous over bismuth quadruple therapy. Furthermore, vonoprazan-based regimens exhibit a lower rate of adverse events than bismuth quadruple therapy.

BACKGROUND AND AIMS:Hemostatic powder (HP) is a novel hemostasis modality for nonvariceal GI bleeding. This meta-analysis was performed to evaluate the efficacy of HP monotherapy versus conventional endoscopic treatment (CET) for nonvariceal GI bleeding. METHODS:PubMed, EMBASE, and Cochrane Library databases were systematically searched from inception to October 16, 2023. The primary outcomes were the initial hemostatic rate and the 30-day recurrent bleeding rate. After the meta-analysis, a trial sequential analysis (TSA) was also conducted to decrease the risk of random errors and validate the result. RESULTS:The meta-analysis included 8 studies, incorporating 653 patients in total. Given significant heterogeneity, all analyses were segregated into malignancy-related and nonmalignancy-related GI bleeding lesions. For the former, HP monotherapy significantly improved the initial hemostasis rate and 30-day recurrent bleeding rate compared with CET (relative risk [RR], 1.50; 95% confidence interval [CI], 1.28-1.75; P < .001; RR, .32; 95% CI, .12-.86; P = .02, respectively), and TSA supported the results. For nonmalignancy-related GI bleeding, HP monotherapy and CET have similar initial hemostasis and 30-day recurrent bleeding rates (RR, 1.08; 95% CI, .98-1.19; P = .11; RR, 1.15; 95% CI, .46-2.90; P = .76, respectively), but the TSA failed to confirm the results. CONCLUSIONS:HP monotherapy surpassed CET in terms of the initial hemostasis rate and 30-day recurrent bleeding rate for patients with malignancy-related GI bleeding. However, their relative efficacy for nonmalignancy-related GI bleeding remains unresolved.

The secretory properties of cancer-associated fibroblasts (CAFs) play predominant roles in shaping a pro-metastatic tumor microenvironment. The present study demonstrated that SLIT2, an axon guidance protein, produced by CAFs and promoted gastric cancer (GC) metastasis in two gastric cancer cell lines (AGS and MKN45) by binding to roundabout guidance receptor 1 (ROBO1). Mass-spectrometry analysis revealed that ROBO1 could interact with NEK9, a serine/threonine kinase. And their mutual binding activities were further enhanced by SLIT2. Domain analysis revealed the kinase domain of NEK9 was critical in its interaction with the intracellular domain (ICD) of ROBO1, and it also directly phosphorylated tripartite motif containing 28 (TRIM28) and cortactin (CTTN) in AGS and MKN45 cells. TRIM28 function as a transcriptional elongation factor, which directly facilitate CTTN activation. In addition, Bioinformatics analysis and experimental validation identified transcriptional regulation of STAT3 and NF-κB p100 by TRIM28, and a synergetic transcription of CTTN by STAT3 and NF-κB p100 was also observed in AGS and MKN45. Therefore, CAF-derived SLIT2 increased the expression and phosphorylation levels of CTTN, which induced cytoskeletal reorganization and GC cells metastasis. A simultaneous increase in the expression levels of NEK9, TRIM28 and CTTN was found in metastatic GC lesions compared with paired non-cancerous tissues and primary cancer lesions via IHC and Multiplex IHC. The analysis of the data from a cohort of patients with GC revealed that increased levels of NEK9, TRIM28 and CTTN were associated with a decreased overall survival rate. On the whole, these findings revealed the connections of CAFs and cancer cells through SLIT2/ROBO1 and inflammatory signaling, and the key molecules involved in this process may serve as potential biomarkers and therapeutic targets for GC.

The characteristics of chemo-sensitive and chemo-resistant GC cell lines. A. Tumors derived from distant metastases were more resistant to chemotherapy. A 3-week 5-FU administration treatment was performed when the secondary model was established for 2 weeks and the tumor burden was then analysed. B-C. The protein expression of HMGA2, FOXL2, ITGA2, E2F1 and Rb in chemo-sensitive and chemo-resistant GC cell lines. **P<0.01. D. The mRNA levels of HMGA2, FOXL2, ITGA2, CDC2, CCNE1 and TK1 in chemo-resistant and chemo-sensitive GC cells. **P<0.01.

HMGA2 regulated cell motility and EMT-like molecular alterations. A. Knockdown of HMGA2 suppressed the migration and invasion of chemo-resistant GC cells in vitro. B. Silencing HMGA2 led to increased E-cadherin and decreased vimentin. **P<0.01. C. Knockdown of HMGA2 suppressed the migration and invasion of chemo-resistant GC cells in vivo. **P<0.01. D. GC cells transfected with HMGA2 acquired enhanced migratory and invasive capacities in vitro. E. Ectopic HMGA2 expression induced EMT-like molecular features in GC cells. **P<0.01. F. HMGA2 promoted cell metastasis in vivo. **P<0.01.

The pink-color sign (PCS) has been widely used for diagnosing esophageal squamous cell carcinoma (ESCC) during Lugol’s iodine chromoendoscopy. However, the identification of the PCS only relies on the subjective assessments made by the endoscopist, which could lead to bias and disagreement. Previous research has indicated that the V′ variable can, as an objective index, define the PCS in the LU′V′ color space. We aimed to validate the diagnostic performance of the PCS defined by the V′ variable alone and attempt to improve the diagnostic performance by combining the V′ and U′ variables. We re-examined 231 subjects with Lugol’s unstained lesions (LULs) from a previously reported prospective trial. The diagnostic performance of the method using V′ variable alone (V′ alone method), the combination method using V′ and U′ variables (V′ + U′ method), and the endoscopists were calculated and compared. A total of 236 LULs were included, among which 46 were histologically confirmed to be cancerous lesions. The sensitivity, specificity, and accuracy of the V′ alone method were 73.91

The expression levels of HMGA2, FOXL2 and ITGA2 are higher in lymph nodes and distant metastases than in primary cancer tissue samples. A. Statistical analysis of the staining intensities of HMGA2, FOXL2, ITGA2 in primary cancer tissue samples and lymph node metastases. B. The comparison of 3 types of GC cases according to the relative expression of 3 proteins between lymph node metastases and primary cancer tissue samples. C. Statistical analysis of the staining intensities of HMGA2, FOXL2, ITGA2 in primary cancer tissue samples and distant metastases. D. The comparison of 3 types of GC cases according to the relative expression of 3 proteins between distant metastases and primary cancer tissue samples.

RT-PCR analysis of HMGA2-FOXL2 pathway associated molecules. A. The mRNA expression levels of E-cadherin, Vimentin, Snail1 and ZEB2 were analysed when FOXL2 was suppressed. **P<0.01. B. The alterations to E-cadherin, Vimentin and Snail1 was analysed after HMGA2 was knocked down. **P<0.01. C. The alterations to E-cadherin, Vimentin, Snail1 and ZEB2 caused by ectopic FOXL2. **P<0.01. D. The effects of ectopic HMGA2 on E-cadherin, Vimentin and Snail1. **P<0.01. E. CDC2, CCNE1 and TK1 were increased by ectopic E2F1 and HMGA2. **P<0.01. F. Knockdown of E2F1 suppressed CDC2, CCNE1 and TK1. **P<0.01. G. CDC2, CCNE1 and TK1 were increased by inhibiting Rb. **P<0.01.

De novo and acquired resistance are major impediments to the efficacy of conventional and targeted cancer therapy. In unselected gastric cancer (GC) patients with advanced disease, trials combining chemotherapy and an anti-EGFR monoclonal antibody have been largely unsuccessful. In an effort to identify biomarkers of resistance so as to better select patients for such trials, we screened the secretome of chemotherapy-treated human GC cell lines. We found that levels of CGA, the α-subunit of glycoprotein hormones, were markedly increased in the conditioned media of chemoresistant GC cells, and CGA immunoreactivity was enhanced in GC tissues that progressed on chemotherapy. CGA levels in plasma increased in GC patients who received chemotherapy, and this increase was correlated with reduced responsiveness to chemotherapy and poor survival. Mechanistically, secreted CGA was found to bind to EGFR and activate EGFR signaling, thereby conferring a survival advantage to GC cells. N-glycosylation of CGA at Asn52 and Asn78 is required for its stability, secretion, and interaction with EGFR. GATA2 was found to activate CGA transcription, whose increase, in turn, induced the expression and phosphorylation of GATA2 in an EGFR-dependent manner, forming a positive feedback circuit that was initiated by GATA2 autoregulation upon sublethal exposure to chemotherapy. Based on this circuit, combination strategies involving anti-EGFR therapies or targeting CGA with microRNAs (miR-708-3p and miR-761) restored chemotherapy sensitivity. These findings identify a clinically actionable CGA/EGFR/GATA2 circuit and highlight CGA as a predictive biomarker and therapeutic target in chemoresistant GC.

Objective: To investigate the relationship between common functional gastrointestinal diseases symptoms with psychological factors, diet and lifestyles by using the network analysis method which has achieved great success in the field of psychology in recent years. Method: A questionnaire survey was conducted in two military units using the cluster sampling method during July 2020, and a total of 1 805 subjects were included. Functional gastrointestinal disease symptoms were evaluated with the Gastrointestinal Symptom Rating Scale (GSRS). The state, trait anxiety scale and stress response scale were used to evaluate the mental and psychological state by self-evaluation. R was used to build the network and calculate statistical parameters. Results: 1 486 of the 1 805 subjects (82.3%) had experienced functional gastrointestinal diseases symptoms within 2 weeks, but most of them were mild. Network analysis shows that there was a strong interaction between digestive system symptoms with different clinical manifestations (Spearman coefficient ranges 0.31-0.56). There was a clear relationship between functional gastrointestinal symptoms and mental and psychological factors (Spearman coefficient ranges 0.16-0.27), but there was no clear interaction with diet, age, education level, body mass index, etc. Functional gastrointestinal diseases symptoms were connected with mental and psychological factors through two nodes: stress and indigestion. The stability coefficient of node strength correlation was 0.75, indicating that the network was stable. Conclusions: The current study revealed the network structure and features of functional gastrointestinal diseases symptoms with mental and psychological factors. The key linking nodes provided potential interfering target for controlling functional gastrointestinal symptoms related to mental and psychological factors.

BACKGROUND The 2016 European Society of Gastrointestinal Endoscopy (ESGE) guidelines recommend that ingested foreign bodies in the upper gastrointestinal (GI) tract are removed as an emergency within 6 hours, with an endoscopic approach that is individualized according to the type of foreign body identified. This retrospective study evaluated the 10-year experience of a single hospital in China performing emergency removal of ingested foreign bodies in 586 adults. MATERIAL AND METHODS Between 2011 and 2020, medical records of 642 adults with a diagnosis of foreign bodies ingestion were retrospectively screened. The timing of endoscopic intervention was classified according to ESGE recommendations. Uni- and multivariate analyses were performed. RESULTS We included 586 patients. The median (range) diameter of foreign bodies was 2.5 (1-24) cm: for sharp ones it was 2.5 (1.5-4.0) cm and for long ones it was 16.9 (10-24) cm. The most common site of foreign body lodgment was the esophagus (n=481; 82.1%); 45.6% (n=252) received emergent removal within 6 hours, while 32.2% (n=178) underwent urgent removal within 24 hours. There were 583 (99.5%) foreign bodies removed successfully and the complication rate was 17.9%. Major complications occurred in 45 patients (7.7%). Female sex and non-emergent endoscopy after 6 hours were significantly associated with a higher overall complications rate. For major complications, older age, time interval >24 hours, and sharper objects were associated with major complications. CONCLUSIONS The findings from this retrospective study support the ESGE statement that endoscopic removal of ingested foreign bodies from the upper GI tract within 6 hours reduces complication rates for adults in the emergency setting.

Rationale: Inflammatory stimuli from the tumor microenvironment play important roles in cancer progression. However, the mechanism of promotion of cancer metastasis by inflammation in gastric cancer (GC) is poorly understood. Methods: The roles of NEK9 were validated via loss-of-function and gain-of-function experiments in vitro and in an animal model of metastasis. Cytoskeletal reorganization-associated molecules were detected by GST pull-down. The regulation of ARHGEF2 by NEK9 was investigated by phosphoproteomics analysis, immunoprecipitation (IP) and in vitro kinase assay. The transcriptional regulation of miR-520f-3p was studied using luciferase reporter and chromatin immunoprecipitation (ChIP). The expression of these proteins in GC tissues was examined by immunohistochemistry. Results: NEK9 directly regulates cell motility and RhoA activation in GC. The phosphorylation of ARHGEF2 by NEK9 is the key step of this process. NEK9 is a direct target of miR-520f-3p, which is transcriptionally suppressed by IL-6-mediated activation of STAT3. A decrease in miR-520f-3p leads to the amplification of IL-6/STAT3 by targeting GP130. A simultaneous elevation of the levels of NEK9, GP130 and p-STAT3 was confirmed in the lymph nodes and distant metastases. An increase in NEK9, GP130 and STAT3 is associated with reduced overall survival of GC patients. Conclusion: This study demonstrates that activation of STAT3 by IL-6 transcriptionally suppresses miR-520f-3p and diminishes the inhibitory effects of miR-520f-3p on NEK9 and GP130. An increase in GP130 enhances this signaling, and NEK9 directly influences cell motility and RhoA activation by targeting the phosphorylation of ARHGEF2. Targeting the IL-6-STAT3-NEK9 pathway may be a new strategy for GC treatment.

Dysbiosis is a prominent feature of inflammatory bowel diseases (IBD). However, the efficacy of using antibiotics aiming at the aberrant gut microflora for IBD treatment are either unsuccessful or not persistent. In contrast, long-term oral vancomycin has been proved effective in controlling both the bile duct and gut inflammation of primary sclerosing cholangitis (PSC), an autoimmune disease against the intraand extrahepatic bile ducts that holds a high rate of concomitant IBD and shares many common characteristics with IBD, including similar dysbiosis patterns. Two discrepancies of antibiotic usage might explain the dramatically different responses of the two diseases toward this strategy. First, the vast majority of antibiotic formulas for IBD management consist of broad-spectrum antibiotics mainly targeting gram-negative bacteria with some covering anaerobes and gram positive ones, while vancomycin used for PSC treatment almost exclusively targets gram-positive bacteria. Several lines of clues suggested that gram-positive microorganisms might be responsible for the chronic inflammation observed in IBD and PSC. Second, improvement of liver test in PSC patients is usually observed after a relatively long period of oral vancomycin treatment (more than 12 weeks) and it takes even longer for gut mucosal healing. Moreover, long-term low dose oral vancomycin is required to prevent PSC recurrence. However, most trials of using antibiotics for IBD management is aiming at inducing remission with short treatment course (most less than 2 weeks) without maintenance. We hypothesize that the host antimicrobial response favors the growth of certain gram-positive intestinal bacteria in genetically predisposed individuals which is responsible for the aberrant immunological reaction towards the gut mucosa. Oral vancomycin induces disease remission by suppressing the pathogenic gram-positive microorganisms, but long course is needed since the gut inflammation is usually severe than that concomitant with PSC. Moreover, long-term maintenance is required to prevent the rebound of the pathogens and flare of the intestinal inflammation.

Aberrant expression of miRNAs is involved in several carcinogenic processes, including tumor growth, metastasis and angiogenesis. The aim of this study was to determine the role of miR-218 in gastric cancer angiogenesis. In situ hybridization was performed on a set of tissue microarray samples to assess the difference in miR-218 expression in vessels between tumor tissues and normal gastric mucosa. In vitro, ectopic expression of miR-218 disturbed the tubular structure and inhibited the migration of endothelial cells. Motility and tube formation were rescued when miR-218 was downregulated. Moreover, miR-218 suppressed endothelial cell sprouting in a fibrin bead sprouting assay. Subsequently, we identified ROBO1 as a target of miR-218 in endothelial cells and determined it was responsible for the effect of miR-218 on tumor angiogenesis. In vivo, local injection of mature miR-218 in xenografted tumors disrupted the vessel plexus and thus inhibited tumor growth. Taken together, our study demonstrated an anti-angiogenic role of miR-218 in gastric cancer and indicated that delivery of miR-218 may be a potential therapeutic strategy to inhibit tumor angiogenesis.

Objective:To explore the role of SLIT2 on the migration ability of different colorectal cancer (CRC) cell lines.Methods:Western blot were performed to detect the expression level of SLIT2 in several CRC cell lines.Based on the expression level of SLIT2,RKO and LOVO were selected for further functional test.siRNA targeting SLIT2 was transfected into RKO,followed by Transwell assay to test the changed migration ability it may induce.LOVO cells transfected with the plasmid containing the coding sequence of SLIT2 were then detected the effect of ectopic SLIT2 on cell migration.Results:SLIT2 silencing facilitated the migration of the RKO.And ectopic expression of SLIT2 distinctly suppressed the wound closure and migration ability of LOVO with high metastasis potential.Conclusion:SLIT2 may exert an inhibitory effect on the migration ability of CRCs.