m6A-related lncRNAs predict prognosis and indicate cell cycle in gastric cancer.

Background: N6-methyladenosine (m6A) modification is a common epigenetic methylation modification of RNA, which plays an important role in gastric carcinogenesis and progression by regulating long non-coding RNA (lncRNA). This study is aimed to investigate the potential prognostic signatures of m6A -related lncRNAs in STAD. Methods: The m6A-related lncRNAs with the most significant impact on gastric cancer prognosis in the TCGA database were identified by bioinformatics and machine learning methods. The m6A-related lncRNA prognostic model (m6A-LPS) and nomogram was constructed by Cox regression analysis with the minimum absolute contraction and selection operator (LASSO) algorithm. The functional enrichment analysis of m6A-related lncRNAs was also investigated. The miRTarBase, miRDB and TargetScan databases were utilized to establish a prognosis-related network of competing endogenous RNA (ceRNA) by bioinformatics methods. The correlation of AL391152.1 expressions and cell cycle were experimentally testified by qRT-PCR and flow cytometry. Results: In total, 697 lncRNAs that were identified as m6A-related lncRNAs in GC samples. The survival analysis showed that 18 lncRNAs demonstrated prognostic values. A risk model with 11 lncRNAs was established by Lasso Cox regression, and can predict the prognosis of GC patients. Cox regression analysis and ROC curve indicated that this lncRNA prediction model was an independent risk factor for survival rates. Functional enrichment analysis and ceRNA network revealed that the nomogram was notably associated with cell cycle. qRT-PCR and flow cytometry revealed that downregulation of GC m6A-related lncRNA AL391152.1 could decrease cyclins expression in SGC7901 cells. Conclusion: A m6A-related lncRNAs prognostic model was established in this study, which can be applied to predict prognosis and cell cycle in gastric cancer.

Esophageal cancer-related gene 4 inhibits gastric cancer growth and metastasis by upregulating Krüppel-like factor 2 expression.

Background: There are a large number of people suffering from gastric cancer (GC) worldwide, so the study of biomarkers for GC is urgently needed. This study aimed to investigate the role of esophageal cancer-related gene 4 (ECRG4) in the growth, metastasis, and prognosis of GC and the possible underlying mechanism. Methods: The expression of ECRG4 was detected in GC tissues by quantitative polymerase chain reaction (PCR), Western blot, and immunohistochemistry. The relationships between ECRG4 expression and clinicopathological parameters of patients with GC were statistically analyzed, and Kaplan-Meier prognosis and survival curves of the patients were plotted. ECRG4 was overexpressed in the human gastric adenocarcinoma cell line (AGS) and human GC cell line 27 (HGC27), and the in vivo effects of ECRG4 overexpression on the growth, invasion, and metastasis of GC were analyzed and verified in nude mice. To identify the downstream transcription factors potentially regulated by ECRG4, ribonucleic acid (RNA) sequencing and differential gene expression analysis were performed on ECRG4-overexpressing cells. Quantitative PCR, Western blot, and immunohistochemistry were used to detect the expression of the downstream transcription factors targeted by ECRG4 in GC. Results: The ECRG4 mRNA and protein expression levels were low in GC tissues and were associated with a poor prognosis. Least absolute shrinkage and selection operator (LASSO) Cox regression and Kaplan-Meier survival analyses showed that patients with low ECRG4 expression had worse prognosis and survival. Overexpression of ECRG4 inhibited the proliferation, metastasis, and invasion of GC cells. RNA sequencing analysis showed that overexpression of ECRG4 induced the upregulation of Krüppel-like factor 2. Conclusions: Our findings show that ECRG4 promotes GC progression via Krüppel-like factor 2 signaling and highlight ECRG4 as a potential GC biomarker and therapeutic target.

Prevalence of antibiotic resistance of Helicobacter pylori isolates in Shanghai, China.

OBJECTIVE: This study aimed to evaluate the resistance of Helicobacter pylori (H. pylori) to common antibiotics in Shanghai. METHODS: A total of 1171 eligible subjects participated in the study. Antibiotic susceptibility to six common antibiotics was examined with the disk diffusion method. Mutations in resistant-related genes were identified via Sanger sequencing analysis. RESULTS: Overall, the resistance rates of strains to amoxicillin, clarithromycin, levofloxacin, metronidazole, tetracycline, and furazolidone were 0.1%, 27.8%, 31.1%, 79.9%, 0.1%, and 0.5%, respectively. Compared with untreated patients, resistance rates of clarithromycin (P < 0.01), levofloxacin (P < 0.01), and metronidazole were significantly higher in re-treated patients (P < 0.05). The total multiple resistance rate was 40.5%. Age (levofloxacin), gender (clarithromycin, levofloxacin, and metronidazole) and endoscopic findings (clarithromycin and levofloxacin) were independent factors influencing antibiotic resistance. High correlation was observed between the drug susceptibility test and molecular test for the resistance to clarithromycin and levofloxacin. CONCLUSIONS: The resistance rates of H. pylori to amoxicillin, tetracycline, and furazolidone were low, whereas the resistance rates of H. pylori to clarithromycin, levofloxacin, and metronidazole were high, especially in re-treated patients. Our results indicate that the clinical resistance patterns of clarithromycin and levofloxacin could be guided by relevant gene mutations.

A study on the diagnosis of the Helicobacter pylori coccoid form with artificial intelligence technology.

Background: Helicobacter pylori (H. pylori) is an important pathogenic microorganism that causes gastric cancer, peptic ulcers and dyspepsia, and infects more than half of the world's population. Eradicating H. pylori is the most effective means to prevent and treat these diseases. H. pylori coccoid form (HPCF) causes refractory H. pylori infection and should be given more attention in infection management. However, manual HPCF recognition on slides is time-consuming and labor-intensive and depends on experienced pathologists; thus, HPCF diagnosis is rarely performed and often overlooked. Therefore, simple HPCF diagnostic methods need to be developed. Materials and methods: We manually labeled 4,547 images from anonymized paraffin-embedded samples in the China Center for H. pylori Molecular Medicine (CCHpMM, Shanghai), followed by training and optimizing the Faster R-CNN and YOLO v5 models to identify HPCF. Mean average precision (mAP) was applied to evaluate and select the model. The artificial intelligence (AI) model interpretation results were compared with those of the pathologists with senior, intermediate, and junior experience levels, using the mean absolute error (MAE) of the coccoid rate as an evaluation metric. Results: For the HPCF detection task, the YOLO v5 model was superior to the Faster R-CNN model (0.688 vs. 0.568, mean average precision, mAP); the optimized YOLO v5 model had a better performance (0.803 mAP). The MAE of the optimized YOLO v5 model (3.25 MAE) was superior to that of junior pathologists (4.14 MAE, p < 0.05), no worse than intermediate pathologists (3.40 MAE, p > 0.05), and equivalent to a senior pathologist (3.07 MAE, p > 0.05). Conclusion: HPCF identification using AI has the advantage of high accuracy and efficiency with the potential to assist or replace pathologists in clinical practice for HPCF identification.

Ferroptosis regulator SLC7A11 is a prognostic marker and correlated with PD-L1 and immune cell infiltration in liver hepatocellular carcinoma.

Background: Liver hepatocellular carcinoma (LIHC) is a complicated disease with poor survival and lack of viable treatment options. The roles of ferroptosis and immunotherapy in LIHC are increasingly prominent, but the interplay of ferroptosis with the tumor microenvironment (TME) in LIHC is currently under-investigated. Methods: In this study, we analyzed normal liver tissues and tumor tissues from the TCGA and GTEx databases to obtain differentially expressed ferroptosis-related genes (FRGs). We then clustered LIHC based on the expression levels of selected FRGs and acquired distinct subtypes with significant heterogeneity regarding survival prognoses, PD-L1 expression, and immune cell infiltration. The correlation of those FRGs with TME in LIHC and pan-cancer analysis was also investigated. GO functional annotations and KEGG pathway analyses were performed to investigate the potential reactions of the obtained differentially expressed genes (DEGs). Further external validation was performed using microarrays on the GEO database and the key ferroptosis regulator SLC7A11 expression between LIHC and normal cells was detected by Western blotting. Results: A large proportion of genes were upregulated in the LIHC group. Among three clusters, cluster 3 had the worst prognosis combined with the highest PD-L1 expression and was positively correlated with various immune cells. Subsequently, survival analysis and Cox regression analysis screened out SLC7A11 as an independent prognostic factor in LIHC featured strong PD-L1 expression and unfavorable survival time. We filter out SLC7A11 as an independent prognostic signature in LIHC patients with strongly associated PD-L1 expression and unfavorable survival probability. In the pan-cancer analysis, high expression of SLC7A11 showed poor overall survival in seven cancers, while the correlation between immune checkpoints (ICs) and SLC7A11 varied by cancer type, indicating the potential therapeutic effects of SLC7A11 in cancers other than LIHC. Western blot was further employed to verify the expression of SLC7A11 in LIHC in vitro. Conclusion: Ferroptosis and TME synergistically play key roles in oncogenesis and progression of LIHC, and SLC7A11 can be used as a predictive biomarker for customized immunotherapy.

INHBB is a novel prognostic biomarker and correlated with immune infiltrates in gastric cancer.

Inhibin subunit beta B (INHBB) is a potential prognostic biomarker for a variety of cancers. However, its role in gastric cancer (GC) remains elusive. The differential expression data of INHBB in tumor and normal tissues were extracted from several databases and genetic alterations of INHBB were assessed by cBioPortal. Kaplan-Meier analysis was used to evaluate the survival rate of patients with GC with INHBB and association with clinical features in GC. Cox regression analysis was used to explore the prognostic value of clinical indicators and INHBB in GC, and a nomogram prognostic model was established. In addition, the predictive validity of the nomogram model was assessed by time-depended receiver operating characteristic (ROC) and calibration curves. Functional enrichment analyses were conducted to functionally annotate INHBB. Notably, we found that the quantitative assessment of immune cell subpopulation infiltration correlated with INHBB expression. INHBB expression is upregulated in GC and is correlated with several clinical features including prognostic indicators and a histological type. Genetic alterations were observed in INHBB, its DNA methylation level was negatively correlated with INHBB expression. High INHBB expression is associated with a poor prognosis and is an independent risk factor for prognosis in GC, along with age and residual tumor. The nomogram model showed a good prediction ability and was validated by time-depended ROC and calibration curves. Functional enrichment analysis indicated that INHBB-associated genes were enriched in tumor microenvironment Gene Ontology (GO) terms and were correlated with tumor-associated pathways. INHBB has a regulatory function in immune cell infiltration, especially macrophage infiltration in GC. Specifically, patients with GC with high INHBB expression and high macrophage infiltration have a worse prognosis. INHBB expression was negatively correlated with the expression of chemokines/chemokine receptors and plays a regulatory role in immunoinhibitor/immunostimulator-involved pathways. INHBB is a potential prognostic biomarker for GC and may drive the abnormal activity of critical cancer-associated pathways, potentially contributing to immune cell infiltration to promote GC development.

A Survey of Helicobacter pylori Antibiotic-Resistant Genotypes and Strain Lineages by Whole-Genome Sequencing in China.

Antibiotic resistance is the most important factor leading to failed Helicobacter pylori eradication therapy, and personalized treatment based on antibiotic susceptibility is becoming increasingly important. To strengthen the understanding of antibiotic genotypic resistance of H. pylori and identify new antibiotic resistance loci, in this study, we identified phenotypic resistance information for 60 clinical isolates and compared the concordance of phenotypic and genotypic resistance using whole-genome sequencing (WGS). Clarithromycin and levofloxacin genotypic resistance was in almost perfect concordance with phenotypic resistance, with kappa coefficients of 0.867 and 0.833, respectively. All strains with the R16H/C mutation and truncation in rdxA were metronidazole resistant, with 100% specificity. For other genes of concern, at least one phenotypically sensitive strain had a previous mutation related to antibiotic resistance. Moreover, we found that the A1378G mutation of HP0399 and the A149G mutation of FabH might contribute to tetracycline resistance and multidrug resistance, respectively. Overall, the inference of resistance to clarithromycin and levofloxacin from genotypic resistance is reliable, and WGS has been very helpful in discovering novel H. pylori resistance loci. In addition, WGS has also enhanced our study of strain lineages, providing new ways to understand resistance information and mechanisms.

Emphasizing the importance of successful eradication of Helicobacter pylori on initial treatment.

Helicobacter pylori (H. pylori) infection is the most important risk factor for gastric cancer and plays an initiating role in the development of intestinal-type gastric cancer. Eradication of H. pylori significantly reduces the incidence and mortality of gastric cancer. International expert consensus recommends eradication treatment for all infected individuals unless competing considerations. However, large-scale H. pylori eradication treatments have led to increasing rates of resistance to multiple antibiotics, together with factors such as coccoid transformation, host CYP2C19 gene polymorphisms, and inappropriate treatment regimens, resulting in a gradual decline in H. pylori eradication rates. Currently, empirical and repeated eradication of H. pylori treatment is common in clinical practice, which will certainly lead to a further increase in antibiotic resistance, resulting in a great waste of medical resources and an increased psychological burden on patients and their relatives. Therefore, successful eradication of H. pylori on initial treatment should be given high priority, and the implementation of personalized treatment is essential.

Duodenal microbiota makes an important impact in functional dyspepsia.

Duodenal microbiota may have impact in Functional Dyspepsia. The aim of this study was to explore the difference of microbiota on duodenal mucosa between patients with Functional Dyspepsia and normal subjects. The duodenal mucosa of the subjects were collected under upper gastrointestinal endoscope and the contents of the descending duodenal intestine were extracted with cell brushes in 20 patients with Functional Dyspepsia and 5 healthy subjects. The microbiome on duodenal was studied by 16SrDNA gene sequencing. The differences of duodenal flora were analyzed and compared by LEfSe, FAPROTAX, SPSS and other software. There were significant differences in ACE index, shannon index and observedspecies index between patients with functional dyspepsia and healthy people (P < 0.05). PCoA analysis of the structure of bacteria between two groups found that the duodenal microbiome showed a separate trend. In further study, Amova analysis showed a significant difference (P < 0.05). We found that the there are obvious differences in the composition of duodenal microbiome in functional dyspepsia and healthy people. At the genus level, there were significant differences in Alloprevotella, Peptostreptococcus,Sutterella, Corynebacteriurn,Catonella, Faecalibacterium,Staphylococcus,Eubacteriumnodatumgro-up, Lachnoclostridiurn and Lautropia between the two groups (P < 0.05). The prediction results of Microflora function from FAPROTAX showed that the urea decomposing (ureolysis) and fumaric acid respiratory (fumaraterespiration) function of duodenal bacteria in patients with functional dyspepsia were significantly different from those in healthy people (P < 0.05). In conclusion, there is a significant difference in mucosal microflora of duodenum between patients with functional dyspepsia and healthy groups. It includes greater microflora diversity, different microflora structure, different microflora composition, specific taxa and specific microbiome function. The disorder of duodenal microecology may be the formation mechanism of functional dyspepsia.

A retrospective study of the antibiotic-resistant phenotypes and genotypes of Helicobacter pylori strains in China.

Helicobacter pylori antibiotic resistance is a serious concern in China, where it severely influences treatment for H. pylori infection. To overcome this, it is essential to apply personalized therapies based on local or individual data on antibiotic-resistant phenotypes or genotypes. We conducted a large-scale multi-center study with a retrospective cross-sectional observational design to investigate the antibiotic-resistant phenotypes and genotypes of H. pylori in China. Strains were isolated from the gastric biopsy samples of H. pylori-infected patients from five different regions in China. The strains were tested for antibiotic-resistant phenotypes and genotypes, and the agreement between the two was assessed. In total, 4242 H. pylori strains were isolated and cultured, with an 84.43% success rate. The primary and secondary antibiotic resistance rates of H. pylori were 37.00% and 76.93% for clarithromycin, 34.21% and 61.58% for levofloxacin, 2.20% and 6.12% for amoxicillin, 1.61% and 3.11% for furazolidone, 1.18% and 3.31% for tetracycline, and 87.87% and 93.48% for metronidazole, respectively. The dual-resistance patterns for metronidazole/clarithromycin, metronidazole/levofloxacin, and clarithromycin/levofloxacin were 43.6%, 38.4%, and 26.1%, respectively. Clarithromycin- and levofloxacin-resistant H. pylori phenotypes and genotypes showed satisfactory agreement. Based on these findings, clarithromycin- and levofloxacin-resistant genotype testing could partially replace traditional antibiotic susceptibility testing in China. Continuous monitoring and personalized treatments based on individual and local H. pylori antibiotic-resistance data remain necessary.

D-galacturonic acid ameliorates the intestinal mucosal permeability and inflammation of functional dyspepsia in rats.

BACKGROUND: Functional dyspepsia (FD) is a gastrointestinal disease caused by imbalanced gastrointestinal function. Traditional treatments are deemed to be limited, and new therapeutic drugs are required. New study suggested that duodenal low-grade inflammation and increased intestinal permeability play an important role in the pathogenesis of FD. Previous studies have shown that polysaccharides containing D-galacturonic acid (GA) could modulate intestinal immune activity in vitro and in animal models. However, the ability of GA monomer to improve intestinal mucosal permeability and inflammation in FD has not been clearly elucidated. METHODS: A FD rat model was established using iodoacetamide (IA). FD Rats were administrated different doses of GA. Subsequently, the body weight and behavioral sensitivity of the rats were measured and evaluated; the permeability of the intestinal barrier was measured by determining D-lactose, lactulose/mannitol ratio (LMR), and permeability-related genes [desmocollin-2 (DSC2), TJP1, and OCLN] in FD rats. Also, inflammatory cells [cluster of differentiation (CD)3+ cells and mast cells] were assessed by immunohistochemistry, and the levels of immune-related factors, such as the Toll-like receptor-nuclear factor kappa B (TLR/NF-κB) pathway, were monitored by reverse transcription quantitative polymerase chain reaction (RT-qPCR) or western blot assays. RESULTS: Our results suggested that GA could markedly increase the body weight and attenuate the behavioral sensitivity of FD rats. Moreover, GA also has an obvious ameliorating effect on the intestinal mucosal permeability and inflammatory response of FD rats. Furthermore, we found that GA could markedly downregulate TLR2, TLR4, and NF-κB in FD rats. CONCLUSIONS: These findings indicate that GA could significantly attenuate the intestinal mucosal permeability and inflammation FD rats. The effect of GA was partially mediated by the TLR/NF-κB signaling pathway.

A case report of a post-polypectomy syndrome with severe sepsis and organ dysfunction.

Post-polypectomy syndrome (PPS) results from electrocoagulation injury to the bowel wall that induces a transmural burn and localized peritonitis. It has a good prognosis; however, there are exceptions when complications are observed. We here report a case of a 50-year-old man who developed lumbosacral pain and high fever with chills four days after colonoscopy, during which polypectomy was performed by endoscopic mucosal resection (EMR) and argon plasma coagulation (APC). Both the plain abdominal film and abdominal CT scan showed no free air, and lumbar CT showed no apparent lesions, which satisfied the diagnosis of PPS. However, the patient was in a critical condition as he developed septic shock caused by bacteremia. Following active treatment, the patient's condition rapidly improved. Therefore, we suggest that clinicians should consider the severity of PPS with sepsis and colon transmural burn. Patients with a diagnosis of PPS should be admitted to the hospital for observation and treatment to avoid adverse consequences.

Low expression of A-kinase anchor protein 5 predicts poor prognosis in non-mucin producing stomach adenocarcinoma based on TCGA data.

BACKGROUND: In the past, there were not a lot of studies on how A-kinase anchor protein 5 (AKAP5) involving in the pathogenesis and prognosis of non-mucin producing stomach adenocarcinoma (NMSA). Therefore, we studied the relationship between AKAP5 and the prognosis of NMSA and its possible mechanisms using publicly available data from The Cancer Genome Atlas (TCGA). METHODS: RNA high-throughput sequencing and clinicopathologic data of NMSA were downloaded from the TCGA. Clinical pathologic features associated with AKAP5 expression were analyzed using the chi-square and Fisher exact tests. The relationship between the overall survival (OS) and AKAP5 expression was analyzed by the Kaplan-Meier method and the Cox regression analysis. GSEA analysis was performed using the TCGA dataset. RESULTS: Our results indicated that the AKAP5 expression was increased in NMSA (all tumor vs. adjacent mucosa). Also, histologic grade, clinical stage, N classification, and survival status were significantly correlated with AKAP5 expression. Kaplan-Meier curves showed that low AKAP5 expression was associated with a poor OS among the NMSA patients (P=5.003e-05), and in the clinical stage III and IV (P=4.646e-05), TNM stage T3 (P=0.016), T4 (P=0.001), N2 (P=0.012), N3 (P=0.003), M0 (P=3.911e-05), and histological grade G3 (P=1.658e-04) subgroups. Cox regression analysis showed that reduced AKAP5 expression in NMSA is associated with age (HR =1.03, P=0.007), stage (HR =1.84 for stage I, II vs. stage III, IV, P=0.002) and M classification (HR =1.8 for M0 vs. M1, P=0.010). Gene sets related to cholesterol homeostasis, glycolysis, estrogen response late, adipogenesis, estrogen response early, notch signaling, and peroxisome were differentially enriched with the low AKAP5 expression phenotype. CONCLUSIONS: Low expression of AKAP5 may be a potential molecular marker for predicting poor prognosis of NMSA. Besides, cholesterol homeostasis, glycolysis, estrogen response, adipogenesis, notch signaling, and peroxisome may be the key pathways regulated by AKAP5 in NMSA. It also suggested that AKAP5 might potentially have biological functions in the development of stomach adenocarcinoma.