Microbial-Related Metabolites May Be Involved in Eight Major Biological Processes and Represent Potential Diagnostic Markers in Gastric Cancer.

Metabolites associated with microbes regulate human immunity, inhibit bacterial colonization, and promote pathogenicity. Integrating microbe and metabolome research in GC provides a direction for understanding the microbe-associated pathophysiological process of metabolic changes and disease occurrence. The present study included 30 GC patients with 30 cancerous tissues and paired non-cancerous tissues (NCs) as controls. LC-MS/MS metabolomics and 16S rRNA sequencing were performed to obtain the metabolic and microbial characteristics. Integrated analysis of the microbes and metabolomes was conducted to explore the coexistence relationship between the microbial and metabolic characteristics of GC and to identify microbial-related metabolite diagnostic markers. The metabolic analysis showed that the overall metabolite distribution differed between the GC tissues and the NC tissues: 25 metabolites were enriched in the NC tissues and 42 metabolites were enriched in the GC tissues. The α and ß microbial diversities were higher in the GC tissues than in the NC tissues, with 11 differential phyla and 52 differential genera. In the correlation and coexistence integrated analysis, 66 differential metabolites were correlated and coexisted, with specific differential microbes. The microbes in the GC tissue likely regulated eight metabolic pathways. In the efficacy evaluation of the microbial-related differential metabolites in the diagnosis of GC, 12 differential metabolites (area under the curve [AUC] >0.9) exerted relatively high diagnostic efficiency, and the combined diagnostic efficacy of 5 to 6 microbial-related differential metabolites was higher than the diagnostic efficacy of a single feature. Therefore, microbial diversity and metabolite distribution differed between the GC tissues and the NC tissues. Microbial-related metabolites may be involved in eight major metabolism-based biological processes in GC and represent potential diagnostic markers.

Leveraging existing 16S rRNA microbial data to identify diagnostic biomarker in Chinese patients with gastric cancer: a systematic meta-analysis.

IMPORTANCE: Gastric cancer is a significant and growing health problem in China. Studies have revealed significant differences in gastric microbiota between patients with gastric cancer and non-cancerous patients, suggesting that microbiota may play a role in tumorigenesis. In this meta-analysis, existing 16S rRNA microbial data were analyzed to find combinations consisting of five genera, which had good efficacy in distinguishing gastric cancer from non-cancerous patients in multiple types of samples. These results lend support to the use of microbial markers in detecting gastric cancer. Moreover, these biomarkers are plausible candidates for further mechanistic research into the role of the microbiota in tumorigenesis.

Antibiotic resistance characteristics and risk factors analysis of Helicobacter pylori strains isolated from patients in Liaoning Province, an area in North China.

Background: The prevalence of Helicobacter pylori (H. pylori) keeps rising while the eradication rate continues to decline due to the increasing antibiotic resistance. Regional variations of antimicrobial resistance to H. pylori have been recommended by guidelines in recent years. This study aims to investigate the antibiotic resistance rate of H. pylori and its association with infected subjects' characteristics in Liaoning Province, an area in north China. Methods: Gastric tissues from 178 H. pylori positive participants without previous antibiotic use within four weeks were collected for H. pylori culture. Antibiotic susceptibility to furazolidone (AOZ), tetracycline (TC), levofloxacin (LFX), metronidazole (MET), clarithromycin (CLA), and amoxicillin (AMX) were examined with the agar dilution method. Associations between H. pylori resistance and patient characteristics were further analysed. Results: No resistance was observed in AOZ or TC. For LFX, MET, CLA, and AMX, the overall resistance rates were 41.10%, 79.14%, 71.78%, and 22.09% respectively. There were significant differences between resistance to CLA and MALToma (P = 0.021), and between resistance to MET and age (P < 0.001). Conclusions: The primary resistant rates of LEX, MET, CLA, and AMX were relatively high in Liaoning. Treatment effectiveness improvement could be achieved by prior antimicrobial susceptibility tests before antibiotic prescription.

The Involvement of TRIB3 and FABP1 and Their Potential Functions in the Dynamic Process of Gastric Cancer.

Background: Dysregulated expression of TRIB3 and FABP1 have been previously observed in human cancer tissues. However, there are little information as to their expression change in dynamic gastric diseases and the functional roles. Methods: Tissues from a total of 479 patients, including 89 GS, 102 IM-GA, 144 EGC, and 144 AGC were collected. The protein expressions of TRIB3 and FABP1 were detected by immunohistochemical staining. Meanwhile, the potential functions of TRIB3 and FABP1 in GC were further analyzed by R software and some internet public databases, such as TCGA and DAVID. Results: During this multi-stage process that go through GS to EGC, the expression trend of TRIB3 and FABP1 protein was GS > IM-GA > EGC. Besides, the expression of TRIB3 protein continued to decrease in AGC, while the expression of FABP1 was abnormally increased. Hp infection was significantly associated with the decreased expression of TRIB3 and FABP1. In addition, the diagnostic efficiency of the combination of these two indicators to diagnose EGC was higher than that of a single indicator. Survival analysis showed that higher expression of TRIB3 or FABP1 could indicate a better prognosis of GC. The protein expressions of TRIB3 and FABP1 were significantly positively correlated. Moreover, CEACAM5 and PRAP1 were positively correlated with both TRIB3 and FABP1 expressions, while GABRP and THBS4 were negatively correlated. The macrophages M0 infiltration was positively correlated with both TRIB3 and FABP1 expressions. Conclusion: The protein expressions of TRIB3 and FABP1 gradually decreased with the gastric disease progress, and was positively correlated. Hp infection may reduce the protein expression of TRIB3 and FABP1. Combing TRIB3 and FABP1 expressions can improve the diagnostic efficiency for EGC. Either a high expression of TRIB3 or FABP1 indicates a better prognosis for GC. TRIB3 and FABP1 may interact with CEACAM5, PRAP1, GABRP and THBS4, and affect tumor immune microenvironment by regulating immune cells, and participate in the development and progression of GC.

A Dynamic Transcriptome Map of Different Tissue Microenvironment Cells Identified During Gastric Cancer Development Using Single-Cell RNA Sequencing.

Gastric cancer (GC) development trends have identified multiple processes ranging from inflammation to carcinogenesis, however, key pathogenic mechanisms remain unclear. Tissue microenvironment (TME) cells are critical for the progression of malignant tumors. Here, we generated a dynamic transcriptome map of various TME cells during multi-disease stages using single-cell sequencing analysis. We observed a set of key transition markers related to TME cell carcinogenic evolution, and delineated landmark dynamic carcinogenic trajectories of these cells. Of these, macrophages, fibroblasts, and endothelial cells exerted considerable effects toward epithelial cells, suggesting these cells may be key TME factors promoting GC occurrence and development. Our results suggest a phenotypic convergence of different TME cell types toward tumor formation processes in GC. We believe our data would pave the way for early GC detection, diagnosis, and treatment therapies.

Interleukin 20 receptor A expression in colorectal cancer and its clinical significance.

BACKGROUND: Interleukin 20 receptor A (IL20RA) has been shown to play a role in the establishment and progression of multiple tumors. However, the expression of this protein in colorectal cancer (CRC) and its correlation with the clinicopathological parameters of CRC have remained unclear. METHODS: A total of 323 paraffin sections including CRC tissues and adjacent normal tissues after surgery were collected. IL20RA protein expression was detected by immunohistochemical staining. The difference expression of IL20RA mRNA between CRC and normal tissues was also explored in the Oncomine and GEO databases. In addition, the IL20RA-related differentially expressed genes were analyzed in TCGA database and enrichment analysis was conducted to explore the cell functions and pathways related to IL20RA expression. RESULTS: There was increased IL20RA expression in CRC compared with that in normal tissues. High IL20RA expression was associated with greater tumor diameter, lymph node metastasis, and poor TNM stage in CRC, while also being suggestive of poor prognosis. The main pathways of IL20RA-related differentially expressed genes in TCGA were protein heterodimerization activity, oxygen binding, oxygen transporter activity, hormone activity, and lipid transporter activity. Meanwhile, IL20RA-related differentially expressed genes were mainly enriched in peroxidase, nucleotide stimulant repair, fatty acid metabolism, basal transcription factor, and RNA degradation. CONCLUSIONS: IL20RA might have a role as a biomarker for CRC. Its upregulation might contribute to an aggressive phenotype in CRC. IL20RA's involvement in the development and progression of CRC might occur through it affecting fatty acid metabolism, oxygen binding, oxygen transport, and hormone activity.

Phenotype characteristics of gastric epithelial mucus in patients with different gastric diseases: from superficial gastritis to gastric cancer.

BACKGROUND: Gastric gland mucin is important for maintaining the basic function of the gastric mucosa, protecting it from foreign substances and reducing the occurrence of gastric diseases. Exploring the phenotype of gastric gland mucus changes during the progression of gastric disease is of great clinical significance. METHODS: A total of 483 patients with different gastric diseases were collected in this study, including 82 superficial gastritis (SG), 81 atrophic gastritis (AG), 168 dysplasia (GD), and 152 gastric cancer (GC). Mucin staining was performed using HID-ABpH2.5-PAS method and was further grouped according to the mucin coloration. RESULTS: The phenotypic characteristics of mucin during disease progression were divided into neutral, acidic, and mucus-free types. Furthermore, acidic mucus can be divided into type I, type II, and type III. The SG group was dominated by neutral mucus (100%), and the AG was dominated by acid mucus (81.48%), which gradually increased with the severity of atrophy (P < 0.05). The GD and GC groups were dominated by mucus-free (43.45%, 78.29%), and as the degree of GD worsened, neutral and acidic mucus gradually decreased and mucus-free increased (P < 0.001). From the SG, AG, GD, and GC progression, neutral and acidic mucus gradually decreased, and mucus- free gradually increased. Acidic mucin revealed that type III (red-brown black) mucin was predominant in AG, GD, and GC, and increased with the degree of AG, GD, as well as the biological behavior of GC. In the lesion adjacent to high-grade GD or GC, type III acid mucin is predominant. CONCLUSION: There were three mucin phenotypes in the process of gastric diseases. With the disease progression, the trend of phenotypic change was that neutral and acidic mucus gradually decreased and mucus-free increased. The appearance of type III mucin suggested a relatively serious phase of gastric diseases and may be a more suitable candidate for follow-up monitoring of patients with GC risk.

Expression of PD1/PDL1 in gastric cancer at different microsatellite status and its correlation with infiltrating immune cells in the tumor microenvironment.

Objective: The microsatellite status and tumor immune microenvironment have a remarkable influence on tumor immunotherapy. This study was performed to investigate programmed cell death protein 1/programmed death ligand 1 (PD1/PDL1) expression and their correlations with CD8+ T cell/CD68+ macrophage (CD68+ M) densities in gastric cancer (GC) at different microsatellite statuses. Methods: The expression of MLH1, PMS2, MSH2, and MSH6 was detected via immunohistochemistry (IHC) to determine the microsatellite status in 215 GC samples obtained from surgical resections. Furthermore, the expression of PD1, PDL1, CD8, and CD68 was detected in the samples via IHC, and the differences and correlations in GC at different microsatellite statuses were then analyzed. PDL1 expression in tumor cells was labeled as PDL1[T], while expression of PD1 and PDL1 in tumor-infiltrating immune cells was labeled as PD1 and PDL1, respectively. Kaplan-Meier analysis was used to evaluate the significance of PD1/PDL1 expression in determining overall survival. Multivariate Cox regression analysis was performed using SPSS software. P-values were determined using the log-rank test. Results: Our results indicated that PD1, PDL1[T], and PDL1 positivity rates were 59%, 35%, and 57% in 46 microsatellite unstable (MSI) GCs and 45%, 22%, and 40% in 169 microsatellite stable (MSS) GCs, respectively. Compared with MSS GC, PD1, PDL1[T], and PDL1 expression was higher in MSI GC (P = 0.109, 0.090, and 0.044, respectively). Additionally, CD8+ T cell and CD68+ M densities were higher in MSI GC than in MSS GC (P = 0.537 and <0.001, respectively). Additionally, CD8+ T cell/CD68+ M densities were evaluated according to tumor center and invasion front. We found that PD1 expression was significantly correlated with CD8+ T cell density at the invasion front of the MSI GC (P = 0.031), whereas PDL1 expression was significantly correlated to high CD68+ M density in the tumor center and invasion front of MSS GC (P = 0.001 and 0.014, respectively). Survival analysis showed that patients with PD1-positive and PDL1[T]/PDL1-negative GC had better prognosis (P = 0.012, 0.005, and 0.022, respectively). Multivariate Cox survival analysis showed that PDL1[T] was an independent prognostic factor for GC. Conclusion: The results suggested that PD1/PDL1 expression and immune response varied at different microsatellite statuses in GC. PD1/PDL1 expression was correlated with CD8+ T cell/CD68+ M densities in GC at different microsatellite statuses, especially at the invasion front. The patients exhibiting high PD1/PDL1 expression or high CD8+ T cell/CD68+ M densities MSI GC might be potential beneficiaries of PD1/PDL1 immunotherapy.

Differentially Expressed mRNAs and Their Long Noncoding RNA Regulatory Network with Helicobacter pylori-Associated Diseases including Atrophic Gastritis and Gastric Cancer.

BACKGROUND: Helicobacter pylori (Hp) infection is the strongest risk factor for gastric cancer (GC). However, the mechanisms of Hp-associated GC remain to be explored. METHODS: The gene expression profiling (GSE111762) data were downloaded from the GEO database. Differentially expressed genes (DEGs) between normal samples (NO) and Hp-atrophic gastritis (GA) or Hp-GA and Hp-GC were identified by GEO2R. Gene Ontology and pathway enrichment analysis were performed using the DAVID database. lncRNA-TF-mRNA and ceRNA regulation networks were constructed using Cytoscape. The cross-networks were obtained by overlapping molecules of the above two networks. GSE27411 and GSE116312 datasets were employed for validation. RESULTS: DEGs between NO and Hp-GA are linked to the activity of inward rectifying potassium channels, digestion, etc. DEGs between Hp-GA and Hp-GC were associated with digestion, positive regulation of cell proliferation, etc. According to the lncRNA-TF-mRNA network, 63 lncRNAs, 12 TFs, and 209 mRNAs were involved in Hp-GA while 16 lncRNAs, 11 TFs, and 92 mRNAs were contained in the Hp-GC network. In terms of the ceRNA network, 120 mRNAs, 18 miRNAs, and 27 lncRNAs were shown in Hp-GA while 72 mRNAs, 8 miRNAs, and 1 lncRNA were included in the Hp-GC network. In the cross-network, we found that immune regulation and differentiation regulation were important in the process of NO-GA. Neuroendocrine regulation was mainly related to the process of GA-GC. In the end, we verified that CDX2 plays an important role in the pathological process of NO to Hp-GA. Comparing Hp-GA with Hp-GC, DEGs (FPR1, TFF2, GAST, SST, FUT9, and SHH), TF, and GATA5 were of great significance. CONCLUSIONS: We identified the DEGs, and their lncRNA regulatory network of Hp-associated diseases might provide insights into the mechanism between Hp infection and GC. Furthermore, in-depth studies of the molecules might be useful to explore the multistep process of gastric diseases.

Identification of cancer stem cell-related biomarkers in intestinal-type and diffuse-type gastric cancer by stemness index and weighted correlation network analysis.

BACKGROUND: Cancer stem cells (CSCs) play an important role in drug resistance, recurrence, and metastasis of tumors. Considering the heterogeneity of tumors, this study aimed to explore the key genes regulating stem cells in intestinal-type and diffuse-type gastric cancer. METHODS: RNA-seq data and related clinical information were downloaded from The Cancer Genome Atlas (TCGA). WGCNA was used to clustered differentially expressed genes with similar expression profiles to form modules. Furtherly, based on the mRNA expression-based stemness index (mRNAsi), significant modules and key genes were identified. Next, the expression of key genes was further verified by the Oncomine database. RESULTS: MRNAsi scores of GC were significantly higher than that of normal tissue. Additionally, mRNAsi scores of intestinal-type GC (IGC) were significantly higher than that of diffuse-type GC (DGC). WGCNA showed that the blue module of IGC and the brown module of DGC were both the most significantly associated with mRNAsi. We screened out 16 and 43 key genes for IGC and DGC and found that these genes were closely related, respectively. Functional analysis showed the relationship between the key genes confirmed in the Oncomine database and the fate of cells. CONCLUSIONS: In this study, 16 and 43 genes related to the characteristics of CSCs were identified in IGC and DGC, respectively. These genes were both associated with cell cycle, which could serve as therapeutic targets for the inhibition of stem cells from both types of GC.

H. pylori slyD, a novel virulence factor, is associated with Wnt pathway protein expression during gastric disease progression.

We have previously reported that the virulence factor HpslyD is related to the occurrence of gastric diseases. However, its mechanism of pathogenesis is still unclear. It is commonly believed that the Wnt/ß-catenin pathway is indispensable for the development of gastric cancer, but it is unclear whether HpslyD and Wnt/ß-catenin interact during the development of gastric diseases. Therefore, we measured the expression of E-cadherin, ß-catenin, TCF4, and CDX2 proteins by IHC in gastric mucosa specimens from patients with different gastric diseases and compared the differences in protein expression to H. pylori-infection status. The results indicated that the expression of these proteins was associated with HpslyD infection. HpslyD subtype infection, rather than common H. pylori infection, may have a greater effect on the expression of Wnt proteins in atrophic gastritis and gastric cancer. Additionally, HpslyD strain infection promoted the expression of Wnt pathway-related proteins with the progression of gastric disease. This study provides insight into the pathogenesis of H. pylori-related gastric diseases and "type-based treatment" for H. pylori infection.

Identification of DEGs and transcription factors involved in H. pylori-associated inflammation and their relevance with gastric cancer.

BACKGROUND: Previous studies have indicated that chronic inflammation linked to H. pylori infection is the leading causes for gastric cancer (GC). However, the exact mechanism is not entirely clear until now. PURPOSE: To identify the key molecules and TFs involved in H. pylori infection and to provide new insights into H. pylori-associated carcinogenesis and lay the groundwork for the prevention of GC. RESULTS: GO and KEGG analysis revealed that the DEGs of Hp+-NAG were mainly associated with the immune response, chemokine activity, extracellular region and rheumatoid arthritis pathway. The DEGs of Hp+-AG-IM were related to the apical plasma membrane, intestinal cholesterol absorption, transporter activity and fat digestion and absorption pathway. In Hp+-NAG network, the expression of TNF, CXCL8, MMP9, CXCL9, CXCL1, CCL20, CTLA4, CXCL2, C3, SAA1 and FOXP3, JUN had statistical significance between normal and cancer in TCGA database. In Hp+-AG-IM network the expression of APOA4, GCG, CYP3A4, XPNPEP2 and FOXP3, JUN were statistically different in the comparison of normal and cancer in TCGA database. FOXP3 were negatively associated with overall survival, and the association for JUN was positive. CONCLUSION: The current study identified key DEGs and their transcriptional regulatory networks involved in H. pylori-associated NAG, AG-IM and GC and found that patients with higher expressed FOXP3 or lower expressed JUN had shorter overall survival time. Our study provided new directions for inflammation-associated oncogenic transformation involved in H. pylori infection.

Modified stepwise mini-incision microdissection testicular sperm extraction: a useful technique for patients with a history of orchidopexy affected by non-obstructive azoospermia.

Non-obstructive azoospermia (NOA), which is defined as the absence of spermatozoa in the ejaculate secondary to impaired spermatogenesis within the testis, may be caused by a variety of etiologies, including varicocele-induced testicular damage, cryptorchidism, prior testicular torsion, post-pubertal mumps orchitis, gonadotoxic effects from medications, genetic abnormalities, chemotherapy/radiation, and other unknown causes currently classified as idiopathic (Cocuzza et al., 2013). The microdissection testicular sperm extraction (micro-TESE) technique involves a meticulous microsurgical exploration of the testicular parenchyma to identify and selectively extract larger seminiferous tubules that carry a higher probability of complete spermatogenesis (Schlegel, 1999). The Cornell group evaluated the efficacy of micro-TESE in 152 NOA patients with an associated history of cryptorchidism. In their series, spermatozoa were successfully retrieved in 116/181 attempts (64%), and the resulting pregnancy rate was 50% with a delivery rate of 38% (Dabaja and Schlegel, 2013). Franco et al. (2016) described a stepwise micro-TESE approach in NOA patients, which was considered to reduce the cost, time, and effort associated with the surgery. Alrabeeah et al. (2016) further reported that a mini-incision micro-TESE, carried through a 1-cm equatorial testicular incision, can be useful for micro-TESE candidates, particularly in patients with cryptozoospermia. We conducted a retrospective study of 20 consecutive NOA patients with a history of orchidopexy from May 2015 to March 2017.

Nucleotide excision repair pathway gene polymorphisms are associated with risk and prognosis of colorectal cancer.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are universally present in nucleotide excision repair (NER) pathway genes, which could make impacts on colorectal carcinogenesis and prognosis. AIM: To explore the association of all tagSNPs in NER pathway genes with colorectal cancer (CRC) risk and prognosis in a northern Chinese population by a two-stage case-control design composed of a discovery and validation stage. METHODS: Genotyping for NER SNPs was performed using kompetitive allele specific PCR. In the discovery stage, 39 tagSNPs in eight genes were genotyped in 368 subjects, including 184 CRC cases and 184 individual-matched controls. In the validation stage, 13 SNPs in six genes were analyzed in a total of 1712 subjects, including 854 CRC cases and 858 CRC-free controls. RESULTS: Two SNPs (XPA rs10817938 and XPC rs2607775) were associated with an increased CRC risk in overall and stratification analyses. Significant cumulative and interaction effects were also demonstrated in the studied SNPs on CRC risk. Another two SNPs (ERCC2 rs1052555 and ERCC5 rs2228959) were newly found to be associated with a poor overall survival of CRC patients. CONCLUSION: Our findings suggest novel SNPs in NER pathway genes that can be predictive for CRC risk and prognosis in a large-scale Chinese population. The present study has referential values for the identification of all-round NER-based genetic biomarkers in predicting the susceptibility and clinical outcome of CRC.

The antibiotic resistance of Helicobacter pylori to five antibiotics and influencing factors in an area of China with a high risk of gastric cancer.

BACKGROUND: H. pylori exhibits antibiotic resistance with regional differences. In this paper, we explored antibiotic resistance of H. pylori to five antibiotics in an area with a high risk of gastric cancer. RESULTS: H. pylori resistance rates to metronidazole, levofloxacin, clarithromycin, amoxicillin, and tetracycline were 78.0, 56.0, 31.0, 9.0, and 15.0%, respectively. Double, triple, quadruple, and quintuple resistance rates were 23, 20, 6, and 4%, respectively. The clarithromycin and multidrug resistance rates were significantly higher in males than females (clarithromycin: 44.4% vs 15.2%, respectively, P = 0.002; multidrug: 75.5% vs 37.2%, respectively; P < 0.001). During the three periods of 1998-1999, 2002-2004 and 2016-2017, the resistance rates to levofloxacin and amoxicillin were increasing (OR: 2.089, 95%CI: 1.142-3.821, P = 0.017; and OR: 5.035, 95%CI: 1.327-19.105, P = 0.018, respectively). The antibiotic resistance rates were unassociated with the host disease state. Metronidazole resistance was lower in the vacAs1m1/m2 group than the vacAs1m1m2 group (65% vs 85.7%, respectively; P = 0.026). As for levofloxacin resistance, it was higher with cagA + than cagA- (60.9% vs 23.1%, respectively; P = 0.020) but lower with slyD+ than slyD- (41.4% vs 68.5%, respectively; P = 0.009). Clarithromycin had a lower resistance rate with iceA++ than iceA-+ (19.7% vs 52.4%, respectively; P = 0.017). For amoxicillin, the iceA++ group had a lower resistance rate than the iceA-- group (1.6% vs 27.8%, respectively; P = 0.009). CONCLUSIONS: The total resistance rates of H. pylori to metronidazole, levofloxacin, clarithromycin, amoxicillin, and tetracycline were high in Zhuanghe. The resistanc rates to levofloxacin and amoxicillin increased over time. Clarithromycin resistance was associated with male and iceA. The resistance of metronidazole was related to vacA. Levofloxacin resistance was concerned with cagA and slyD and amoxicillin resistance was concerned with iceA. While, the antibiotic resistance of H. pylori had nothing to do with the status of gastric disease.

Mucosa-Associated Microbiota in Gastric Cancer Tissues Compared With Non-cancer Tissues.

The link between microbiota and gastric cancer (GC) has attracted widespread attention. However, the phylogenetic profiles of niche-specific microbiota in the tumor microenvironment is still unclear. Here, mucosa-associated microorganisms from 62 pairs of matched GC tissues and adjacent non-cancerous tissues were characterized by 16S rRNA gene sequencing. Functional profiles of the microbiota were predicted using PICRUSt, and a co-occurrence network was constructed to analyze interactions among gastric microbiota. Results demonstrated that mucosa-associated microbiota from cancerous and non-cancerous tissues established micro-ecological systems that differed in composition, structure, interaction networks, and functions. Microbial richness and diversity were increased in cancerous tissues, with the co-occurrence network exhibiting greater complexity compared with that in non-cancerous tissue. The bacterial taxa enriched in the cancer samples were predominantly represented by oral bacteria (such as Peptostreptococcus, Streptococcus, and Fusobacterium), while lactic acid-producing bacteria (such as Lactococcus lactis and Lactobacillus brevis) were more abundant in adjacent non-tumor tissues. Colonization by Helicobacter pylori, which is a GC risk factor, also impacted the structure of the microbiota. Enhanced bacterial purine metabolism, carbohydrate metabolism and denitrification functions were predicted in the cancer associated microbial communities, which was consistent with the increased energy metabolism and concentration of nitrogen-containing compounds in the tumor microenvironment. Furthermore, the microbial co-occurrence networks in cancerous and non-cancerous tissues of GC patients were described for the first time. And differential taxa and functions between the two groups were identified. Changes in the abundance of certain bacterial taxa, especially oral microbiota, may play a role in the maintenance of the local microenvironment, which is associated with the development or progression of GC.

Seminiferous tubule molecular imaging for evaluation of male fertility: Seeing is believing.

The proper assessment of male fertility is essential for diagnosing and treating male infertility. Currently, spermiogram and Johnsen testicular biopsy score counts are used to assess male fertility. However, spermiogram is not a suitable option for non-obstructive azoospermia patients, and Johnsen testicular biopsy scores only represent localized and not the overall spermatogenesis. Whole-mount staining was a novel method for evaluating protein expression in the tissue. Thus, we explored its application in human seminiferous tubules. Testicular biopsies from 57 azoospermia patients were categorized as obstructive azoospermia (OA), maturation arrest (MA) and Sertoli-cells only syndrome (SCOS). We performed whole-mount staining of their seminiferous tubules and evaluated the spermatogonial stem cells (SSCs), differentiated spermatogonia (SG), spermatocytes (SPC) and spermatids (SD) with their respective markers (GFRA1, CD117, SYCP3, and PNA) to assess fertility. GFRA1, CD117, SYCP3, and PNA were not expressed in SCOS patients, whereas all of them were detected in OA patients. In MA patients with arrested spermatogenesis at the SPC stage, GFRA1, CD117, and SYCP3, but not PNA were expressed in the seminiferous tubules. In MA patients with arrested spermatogenesis at the spermatogonia stage, only GFRA1 was expressed in the seminiferous tubules. These results were consistent with the Johnsen testicular biopsy score counts except for one patient, where although only Sertoli cells were indicated by the score, SSCs were also detected in the whole-mounts. Collectively, whole-mount staining could be used to analyze the inherent spermatogenesis of seminiferous tubules through staining of germ cells at different stages. It offers a more accurate and promising faster method for assessing male fertility compared with traditional biopsy screening. And it could have potential value for the clinical purpose for male fertility management.

Comprehensive Analysis of Aberrantly Expressed ceRNA network in gastric cancer with and without H.pylori infection.

Objective: This study mainly focused on revealing ceRNA network in gastric cancer (GC) with Hp infection after comparing with GC without Hp infection and exploring the biological function and prognostic relevance of related molecules. Methods: The RNA expression profile data of GC patients with or without Hp infection were extracted from TCGA GDC data portal, including 20 GC cases with Hp infection and 168 GC cases without Hp infection. Differentially expressed lncRNAs, miRNAs and mRNAs were unveiled by package edgeR of R, and lncRNA-miRNA-mRNA ceRNA network was constructed by integrating the miRNA target information and the expression data of lncRNAs, miRNAs and mRNAs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of aberrantly expressed mRNAs were performed to identify the related biological functions and pathologic pathways, and protein-protein interaction (PPI) network was constructed by STRING database. The overall survival (OS) of aberrantly expression lncRNAs and miRNAs were analyzed by package survival of R. A total of 30 gastric cancer tissues were used to validate the bioinformatics analysis results by real-time PCR. Results: Among the 32 differentially expressed miRNAs, 27 differentially expressed lncRNAs and 257 differentially expressed mRNAs were identified by comparing GC patients with and without Hp infection. Totally 10 miRNA, 11 lncRNA, 219 mRNA were included to build ceRNA network. GO and KEGG analysis revealed that differentially expressed mRNAs involved in the ceRNA network were mainly involved in extracellular exosomes, structural molecular activities, proteolysis and P13K-Akt signaling pathways. And PPI analysis obtained six hub genes of NTS, APOC3, OTX2, KRT13, CALCA, GNG4. Survival analysis showed that four lncRNAs (LINC01254, LINC01287, LINC01524, U95743.1) and four miRNAs (miR-302a, miR-302b, miR-1286, miR-378g) were associated with overall survival of GC with Hp infection. The real-time PCR results showed that, the levels of LINCO1254, LINCO1287, LINCO1524, U95743.1 were significantly higher in Hp positive GC patients than Hp negative patients (P=0.02, 0.048, 0.04, 0.036, respectively). Conclusion: Using TCGA database for data mining, we have successfully constructed a ceRNA regulatory network of GC with Hp infection, consisting of 10 lncRNAs, 11 miRNAs and 219 mRNAs. These findings might provide critical clues for the regulatory role of ceRNA network in the development of GC with Hp infection.

Role of abnormal microRNA expression in Helicobacter pylori associated gastric cancer.

Epidemiological studies have shown that Helicobacter pylori (HP) infection is a risk factor for gastric cancer (GC). HP infection may induce the release of pro-inflammatory mediators, and abnormally increase the level of reactive oxygen species (ROS), nitric oxide (NO), and cytokines in mucosal epithelial cells of the stomach. However, the specific mechanism underlying the pathogenesis of HP-associated GC is still poorly understood. Recent studies have revealed that abnormal microRNA expression may affect the proliferation, differentiation, and apoptosis of mucosal epithelial cells of the stomach to further influence GC occurrence, development, and metastasis. Herein, we summarize the role of abnormal microRNAs in the regulation of HP-associated GC progression. Abnormal microRNA expression in HP-positive GC may be a biomarker for GC diagnosis, occurrence, and development as well as its targeted treatment and prognosis.

Label-free detection of pepsinogen 1 and 2 by polyethylene coating Lamb microfluidic device.

Early screening of gastric cancer is a critical importance for the improvement of patients' survival rate. Here, a polyethylene coating Lamb (PE-Lamb) microfluidic device with immune layer for gastric cancer label-free detection was constructed. Two serum pepsinogen 1 (PG1) and pepsinogen 2 (PG2) biomarkers were applied to screen and predict the appearance of gastric cancer. Compared with enzyme-linked immunosorbent assay (ELISA), this method achieved a higher sensitivity and less time (40 min vs 120 min). The limit of detections (LOD) were reached 60 pg/mL for PG1 and 30 pg/mL for PG2, which have two orders of magnitude lower than traditional ELISA. The linearity coefficient indexes (R2) for PG1 and PG2 were 0.992 and 0.953 respectively, which is similar to that of ELISA. In addition, PG1 and PG2 mixed antigens sample with human serum was detected by PE-Lamb approach, and the frequency response showed high reproducibility and specificity. The results indicate that PE-lamb diagnostic technique is a novel and promising method for high-throughput screening and early diagnosis of gastric cancer.