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The mucosa-associated microbiota is widely recognized as a potential trigger for Crohn’s disease pathophysiology but remains largely uncharacterised beyond its taxonomic composition. Unlike stool microbiota, the functional characterisation of these communities using current DNA/RNA sequencing approaches remains constrained by the relatively small microbial density on tissue, and the overwhelming amount of human DNA recovered during sample preparation. Here, we have used a novel ex vivo approach that combines microbe culture from anaerobically preserved tissue with metagenome sequencing (MC-MGS) to reveal patient-specific and strain-level differences among these communities in post-operative Crohn’s disease patients. The 16?S rRNA gene amplicon profiles showed these cultures provide a representative and holistic representation of the mucosa-associated microbiota, and MC-MGS produced both high quality metagenome-assembled genomes of recovered novel bacterial lineages. The MC-MGS approach also produced a strain-level resolution of key Enterobacteriaceaand their associated virulence factors and revealed that urease activity underpins a key and diverse metabolic guild in these communities, which was confirmed by culture-based studies with axenic cultures. Collectively, these findings using MC-MGS show that the Crohn’s disease mucosa-associated microbiota possesses taxonomic and functional attributes that are highly individualistic, borne at least in part by novel bacterial lineages not readily isolated or characterised from stool samples using current sequencing approaches.

The mucosa-associated microbiota is widely recognized as a potential trigger for Crohn’s disease pathophysiology but remains largely uncharacterised beyond its taxonomic composition. Unlike stool microbiota, the functional characterisation of these communities using current DNA/RNA sequencing approaches remains constrained by the relatively small microbial density on tissue, and the overwhelming amount of human DNA recovered during sample preparation. Here, we have used a novel ex vivo approach that combines microbe culture from anaerobically preserved tissue with metagenome sequencing (MC-MGS) to reveal patient-specific and strain-level differences among these communities in post-operative Crohn’s disease patients. The 16 S rRNA gene amplicon profiles showed these cultures provide a representative and holistic representation of the mucosa-associated microbiota, and MC-MGS produced both high quality metagenome-assembled genomes of recovered novel bacterial lineages. The MC-MGS approach also produced a strain-level resolution of key Enterobacteriaceaand their associated virulence factors and revealed that urease activity underpins a key and diverse metabolic guild in these communities, which was confirmed by culture-based studies with axenic cultures. Collectively, these findings using MC-MGS show that the Crohn’s disease mucosa-associated microbiota possesses taxonomic and functional attributes that are highly individualistic, borne at least in part by novel bacterial lineages not readily isolated or characterised from stool samples using current sequencing approaches.

REC8 meiotic recombination protein (REC8) was found to be preferentially methylated in gastric cancer (GC) using promoter methylation array. We aimed to elucidate the epigenetic alteration and biological function of REC8 in GC. REC8 was downregulated in 100% (3/3) of Epstein–Barr virus (EBV)-positive and 80% (8/10) of EBV-negative GC cell lines by promoter methylation, but the expression could be restored through demethylation treatment. Protein expression of REC8 was significantly lower in human primary gastric tumors than in adjacent non-tumor tissues. A negative correlation between methylation and mRNA expression of REC8 was observed in 223 gastric samples of The Cancer Genome Atlas study (r=−0.7018, P<0.001). The methylation level (%) of the REC8 promoter was significantly higher in EBV-positive gastric tumors than in EBV-negative gastric tumors, as shown by bisulfite genomic sequencing (77.6 (69.3–80.5) vs 51.4 (39.5–62.3), median (interquartile range); P<0.001); methylation levels in both subtypes of tumors were significantly higher than in normal stomach tissues (14.8 (4.2–24.0)) (both P<0.001). Multivariate analysis revealed that REC8 methylation was an independent factor for poor survival in GC patients (hazard ratio=1.68, P<0.05). REC8 expression significantly suppressed cell viability, clonogenicity and cell cycle progression; it induced apoptosis and inhibited migration of AGS-EBV (EBV-positive) and BGC823 (EBV-negative) GC cells, and it suppressed tumorigenicity in nude mice. In contrast, knockdown of REC8 in gastric epithelial immortalized GES-1 cells significantly increased cell viability, clonogenicity and migration ability. The tumor-suppressive effect of REC8 is mediated at least in part by the downregulation of genes involved in cell growth (G6PD, SLC2A1, NOL3, MCM2, SNAI1 and SNAI2), and the upregulation of apoptosis/migration inhibitors (GADD45G and LDHA) and tumor suppressors (PinX1, IGFBP3 and ETS2). In conclusion, REC8 is a novel tumor suppressor that is commonly downregulated by promoter methylation in GC, especially in the EBV-associated subtype. Promoter methylation of REC8 is an independent risk factor for the shortened survival of GC patients.

Epidemiological studies showed that obesity and its related non-alcoholic fatty liver disease (NAFLD) promote hepatocellular carcinoma (HCC) development. We aimed to uncover the genetic alterations of NAFLD-HCC using whole-exome sequencing. We compared HCC development in genetically obese mice and dietary obese mice with wild-type lean mice fed a normal chow after treatment with diethylnitrosamine. HCC tumor and adjacent normal samples from obese and lean mice were then subjected to whole-exome sequencing. Functional and mechanistic importance of the identified mutations in Carboxyl ester lipase (Cel) gene and Harvey rat sarcoma virus oncogene 1 (Hras) was further elucidated. We demonstrated significantly higher incidences of HCC in both genetic and dietary obese mice with NAFLD development as compared with lean mice without NAFLD. The mutational signatures of NAFLD-HCC and lean HCC were distinct, with <3% overlapped. Eight metabolic or oncogenic pathways were found to be significantly enriched by mutated genes in NAFLD-HCC, but only two of these pathways were dysregulated by mutations in lean HCC. In particular, Cel was mutated significantly more frequently in NAFLD-HCC than in lean HCC. The multiple-site mutations in Cel are loss-of-function mutations, with effects similar to Cel knock-down. Mutant Cel caused accumulation of cholesteryl ester in liver cells, which led to induction of endoplasmic reticulum stress and consequently activated the IRE1α/c-Jun N-terminal kinase (JNK)/c-Jun/activating protein-1 (AP-1) signaling cascade to promote liver cell growth. In addition, single-site mutations in Hras at codon 61 were found in NAFLD-HCC but none in lean HCC. The gain-of-function mutations in Hras (Q61R and Q61K) significantly promoted liver cell growth through activating the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/3-phosphoinositide-dependent protein kinase-1 (PDK1)/Akt pathways. In conclusion, we have identified mutation signature and pathways in NAFLD-associated HCC. Mutations in Cel and Hras have important roles in NAFLD-associated hepatocellular carcinogenesis.

Nude mice are important in vivomodel for characterization of cell malignancy behavior; however, many cancer cells fail to form tumors in it. Understanding this defective mechanism may provide novel insights into tumorigenesis and how tumor cells escape innate immunity. Whole-genome sequencing was conducted on two gastric cancer (GC) cells, BGC823 and AGS, which do and do not form tumors in nude mice, to identify their genomic differences relevant to natural killer (NK) cells. We found that the tumorigenic capacity of human GC cell lines was dependent on the recruitment and activation of NK cells in xenograft tumors. We used whole-genome sequence (WGS) on GC cell lines to identify potential genes controlling susceptibility to NK-mediated killing. The tumorigenic cell line BGC823 expressed high levels of HLA-I because of copy gain and was resistant to NK cell killing. In contrast, another cell line AGS expressing low levels of HLA-I with activated NKp30/MAPK/IL-12 (interleukin-12) or IL-2 (interleukin-2) pathway was susceptible to NK lysis. Treatment of tumor bearing mice with systemic administration of IL-12 in combination with intratumor injection of anti-HLA-I antibody significantly increased NK cell recruitment into xenograft tumors, which became sensitive to NK killing, resulting in reduced tumor progression. In human GC specimens, decreased HLA-I expression and increased NK cells surrounding tumor cells were correlated with decreased metastasis potential and better prognosis of patients. Our results provide a mechanistic basis for GC cells to escape NK lysis and a promising prospect of NK immunotherapy for GC cells.

The Ras association domain family (RASSF) encodes several members with tumor-suppressive potentials. We aimed to investigate the biological function and clinical implication of RASSF10 in gastric cancer (GC). We found that RASSF10was silenced in six of seven GC cell lines and in primary GC tissues, but was highly expressed in normal gastric tissues. The silence of RASSAF10 was mediated by promoter methylation as evaluated by bisulfite genomic sequencing. RASSF10expression could be restored by demethylation treatment. A negative correlation between methylation and mRNA expression of RASSF10was observed in 223 gastric samples of The Cancer Genome Atlas study (P<0.0001). Re-expression of RASSF10 in GC cell lines (AGS and MKN45) significantly suppressed cell viability, colony formation, migration and invasion, reduced cells in S phase, accumulated cells in G2 phase and induced cell apoptosis in vitro, and inhibited tumorigenicity in nude mice. These were confirmed by decreased expression of proliferation markers (proliferating cell nuclear antigen, p-CDC2 and p-CDC25) and increased apoptotic cascades (cleaved caspases-9, -8, -3 and cleaved poly (ADP-ribose) polymerase). Conversely, RASSF10knockdown in normal gastric cell line yielded an opposing effect. Co-immunoprecipitation combined with mass spectrometry analyses were performed to reveal the downstream effectors of RASSF10. The result revealed that glutathione S-transferase Pi 1 (GSTP1) was a direct cooperator of RASSF10. The tumor-suppressive effect of RASSF10 was partially mediated by cooperating with GSTP1 to deregulate Jun N-terminal kinase (JNK)/c-Jun/AP-1 pathway. Importantly, RASSF10methylation was detected in 56.6% (98/173) of primary GCs and is an independent risk factor for poor survival of GC patients (P=0.001). In conclusions, RASSF10 functions as a tumor suppressor by cooperating with GSTP1 to deregulate JNK/c-Jun/AP-1 pathway in GC. Promoter methylation of RASSF10is associated with poor survival of GC patients.

Accumulating evidence reveals the effectiveness of epigenetic therapy in gastric cancer. However, the molecular mechanisms and targets underlying such therapeutic responses remain elusive. Herein, we report an aberrant yet therapeutically rectifiable epigenetic signaling in gastric carcinogenesis. Administration of DNA-demethylating drug 5-aza-2'-deoxycytidine (5-aza-dC) reduced gastric cancer incidence by ~74% (P<0.05) in N-nitroso-N-methylurea-treated mice. Through genome-wide methylation scanning, novel promoter hypermethylation-silenced and drug-targeted genes were identified in the resected murine stomach tumors and tissues. We uncovered that growth/differentiation factor 1(Gdf1), a member of the transforming growth factor-ß superfamily, was silenced by promoter hypermethylation in control tumor-bearing mice, but became reactivated in 5-aza-dC-treated mice (P<0.05). In parallel, the downregulated SMAD2/3 phosphorylation in gastric cancer was revived by 5-aza-dC in vivo. Such hypermethylation-dependent silencing and 5-aza-dC-mediated reactivation of GDF1-SMAD2/3 activity was conserved in human gastric cancer cells (P<0.05). Subsequent functional characterization further revealed the antiproliferative activity of GDF1, which was exerted through activation of SMAD2/3/4-mediated signaling, transcriptional controls on p15, p21and c-Myccell-cycle regulators and phosphorylation of retinoblastoma protein. Clinically, hypermethylation and loss of GDF1was significantly associated with reduced phosphorylated-SMAD2/3 and poor survival in stomach cancer patients (P<0.05). Taken together, we demonstrated a causal relationship between DNA methylation and a tumor-suppressive pathway in gastric cancer. Epigenetic silencing of GDF1abrogates the growth-inhibitory SMAD signaling and renders proliferation advantage to gastric epithelial cells during carcinogenesis. This study lends support to epigenetic therapy for gastric cancer chemoprevention and identifies a potential biomarker for prognosis.

Whole-genome and transcriptome sequencing were used to discover novel gene fusions in a case of colon cancer. A tumor-specific LACTB2-NCOA2 fusion originating from intra-chromosomal rearrangement of chromosome 8 was identified at both DNA and RNA levels. Unlike conventional oncogenic chimeric proteins, the fusion product lacks functional domain from respective genes, indicative of an amorphic rearrangement. This chimeric LACTB2-NCOA2 transcript was detected in 6 out of 99 (6.1%) colorectal cancer (CRC) cases, where NCOA2 was significantly downregulated. Enforced expression of wild-type NCOA2 but not the LACTB2-NCOA2 fusion protein impaired the pro-tumorigenic phenotypes of CRC cells, whereas knockdown of endogenous NCOA2 in normal colonocytes had opposite effects. Mechanistically, NCOA2 inhibited Wnt/β-catenin signaling through simultaneously upregulating inhibitors and downregulating stimulators of Wnt/β-catenin pathway. Collectively, our data supports that NCOA2 is a novel negative growth regulatory gene repressing the Wnt/β-catenin pathway in CRC, where recurrent fusion with LACTB2 contributes to its disruption.

A well-described animal model was used to understand the molecular mechanisms of carcinogenesis and metastasis of gastric cancer at the protein level. Gastric cancer was induced in 12 Wistar rats by oral administration of N-methyl-N‘-Nitro-N−Nitrosoguanidine (MNNG). The protein expression patterns of normal gastric tissue, gastric cancer, and corresponding metastases were analyzed by proteomics in matched tissues of 3 rats. Proteins in the region of molecular masses of 15−75 kDa and an isoelectric point of 3−7 were separated by two-dimensional electrophoresis (2-DE) and identified by peptide fingerprinting with matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF−MS). Twenty-seven spots corresponding to 25 different proteins served as landmarks for comparison between tissues. The identified proteins included cytoskeletal proteins, stress associated proteins, proteins involved in signal transduction, cell proliferation and differentiation, and metabolism. Eleven proteins were up-regulated and 2 proteins were down-regulated in tumor tissue when compared with normal tissue. Twelve proteins were up-regulated and 8 proteins were down-regulated in the metastases when compared with the primary tumor. The overexpression of HSP27 in gastric cancer was confirmed by immunohistochemical analysis of human gastric cancer specimens. Combining well-defined animal models with proteome analysis will improve our understanding of the fundamental changes that contribute to the process of carcinogenesis and the formation of metastases in gastric cancer. Keywords: gastric cancer • metastasis • proteome analysis • two-dimensional electrophoresis • matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry

Severe acute respiratory syndrome (SARS) struck Hong Kong bitterly in the spring of 2003, infecting 1755 persons and claiming nearly 300 lives. The epidemic was introduced by travellers from southern China, where the disease had originated. It started in late February and lasted until early June. Two notable ‘super-spreading’ events were reported, one inside a teaching hospital and the other in a private housing estate. Other than in the super-spreading events, the infectivity in the community appeared to be low, and there were few, if any, asymptomatic or subclinical infections. Health-care workers were at particular risk and accounted for 22% of all probable cases. The main modes of transmission were through droplet spread and close/direct contacts, but situations conducive to aerosol generation appeared to be associated with higher risk. Our review suggests that there are still many unknown factors concerning the mode of transmission and environmental risk that need to be clarified.

BACKGROUND: Continued or recurrent bleeding after endoscopic treatment for bleeding ulcer is a major adverse prognostic factor. Identification of such ulcers may allow for alternate treatments. AIM: To determine factors predicting treatment failure with combined adrenaline injection and heater probe thermocoagulation. Methods: Consecutive patients with bleeding peptic ulcers who received endoscopic therapy between January 1995 and March 1998 were studied. Data on clinical presentation, endoscopic findings, and treatment outcomes were collected prospectively. Multiple logistic regression analysis was used to identify independent risk factors for treatment failure. RESULTS: During the study period, 3386 patients were admitted with bleeding peptic ulcers: 1144 (796 men, 348 women) with a mean age of 62.5 (SD 17.6) years required endoscopic treatment. There were 666 duodenal ulcers (58.2%), 425 gastric ulcers (37.2%), and 53 anastomotic ulcers (4.6%). Initial haemostasis was successful in 1128 patients (98.6%). Among them, 94 (8.2%) rebled in a median time of 48 hours (range 3-480). Overall failure rate was 9.6%. Mortality rate was 5% (57/1144). Multiple logistic regression analysis revealed that hypotension (odds ratio (OR) 2.21, 95% confidence interval (CI) 1.40-3.48), haemoglobin level less that 10 g/dl (OR 1.87, 95% CI 1.18-2.96), fresh blood in the stomach (OR 2.15, 95% CI 1.40-3.31), ulcer with active bleeding (OR 1.65, 95% CI 1.07-2.56), and large ulcers (OR 1.80, 95% CI 1.15-2.83) were independent factors predicting rebleeding. CONCLUSIONS: Larger ulcers with severe bleeding at presentation predict failure of endoscopic therapy.

Helicobacter pylori synthesizes an unusual GroES homolog, heat shock protein A (HspA). The present study was aimed at an assessment of the serological response to HspA in a group of Chinese patients with defined gastroduodenal pathologies and determination of whether diversity is present in the nucleotide sequences encoding HspA in isolates from these patients. Serum samples collected from 154 patients who had an upper gastrointestinal pathology and the presence of H. pylori defined by biopsy were tested for an immunoglobulin G (IgG) serologic response to H. pylori HspA by an enzyme linked immunosorbant assay. HspA-encoding nucleotide sequences in H. pylori isolates from 14 patients (7 seropositive and 7 seronegative for HspA) were analyzed by PCR and direct sequencing of the PCR products. The sequencing results were compared to those of 48 isolates from other parts of the world. Of the 154 known H. pylori-positive patients, 54 (35.1%) were seropositive for HspA. The A domain (GroES homology) of HspA was highly conserved in the 14 isolates tested. Although the B domain (metal-binding site unique to H. pylori) resembled that in the known major variant, particular amino acid substitutions allowed definition of an HspA variant associated with isolates from East Asia. There were no associations between patient characteristics and HspA seropositivity or amino acid sequences. We confirmed in this study that the clinical outcomes of H. pylori infection are not related to HspA antigenicity or to sequence variation. However, B-domain sequence variation may be a marker for the study of the genetic diversity of H. pylori strains of different geographic origins.

Barium enema and colonoscopy are commonly used for the investigation of suspected colonic cancer. These techniques are relatively invasive and both the investigation and the preceding bowel preparation are demanding, particularly in the elderly. A prospective, blinded trial was conducted to compare ultrasound (US) and CT with colonoscopy. CT and colonoscopy were performed on 50 patients with symptoms suggesting colonic cancer. Both radiological investigations were performed prior to the bowel preparation for colonoscopy. US was performed without any preparation and oral contrast medium was the only preparation used for CT. Colonoscopy detected six cancers, all of which were diagnosed by both US and CT. In addition, US and CT diagnosed a further cancer not seen on colonoscopy due to an incomplete study. US had a sensitivity and specificity of 100% and CT a sensitivity of 100% and a specificity of 84%. US and CT were poor at diagnosing polyps. If the detection of polyps greater than 2 cm is included then US sensitivity falls to 67% and CT sensitivity falls to 89% and specificity rises to 88%. In conclusion, both US and CT are possible alternatives to colonoscopy in the investigation of symptomatic patients with suspected colonic cancer. The use of these techniques could markedly reduce the need for colonoscopy in this patient population with attendant cost savings. Non-invasive imaging has particular advantages in the elderly who cope poorly with both the bowel preparation and the procedure.

To identify the risk factors for developing recurrent ulcer complications after recovery from an episode of peptic ulcer bleeding 611 patients admitted with peptic ulcer bleeding were studied. Some 557 (91 per cent) were discharged without operation. A total of 22 patients were lost to follow-up and five were excluded as maintenance H2blockers were required. Of the remaining 530 patients at risk, 169 (32 per cent) developed another complication (166 bleeding, three perforations) over a median follow-up period of 36 months. Patients with duodenal ulcers at the time of bleeding, previous history of peptic ulcer, previous bleeding, history of dyspepsia longer than 3 months, and a short interval between previous ulcer complications and the index bleed were more likely to develop further complications. Sex, age, smoking, coexisting illness, non-steroidal anti-inflammatory drugs intake and time taken to achieve ulcer healing had no predictive value.

The objective was to study the gastrointestinal complications in chronic NSAID users with Helicobacter pylori infection. Eighty-two Chinese patients on long-term NSAID or aspirin treatment were studied for the occurrence of H. pylori infection and gastroduodenal mucosal injuries by upper endoscopy. H. pylori infection was confirmed by CLO test, histology and bacteriological cultures. Frequency and severity of symptoms of dyspepsia were also assessed.

In the last decade, endoscopic haemostasis, including injection sclerotherapy and variceal ligation has become the standard method for the treatment of variceal haemorrhage. While the use of vasopressin has been hampered by severe side effects, the development of new vasoactive agents such as terlipressin, somatostatin and octreotide has opened new indications for these pharmacological therapies. These agents are effective, safe and easy to use. They could be recommended as stop‐gap treatment before endoscopy can be arranged. They can also be used as adjunct therapy for endoscopic haemostasis in acute variceal haemorrhage.

Endoscopic biliary stenting has become a standard palliative treatment for obstructive jaundice due to malignancies of the pancreas and the hepatobiliary system. Despite the high initial success rate in achieving biliary drainage, durable endoscopic stenting has been limited by the clogging of biliary stents, usually after 4–5 months, due to formation of an adherent bacterial biofilm. Various methods have been investigated for the prevention of bacterial adhesion and prolongation of stent patency. These include: 1) prophylactic use of antimicrobial agents and bile salts; 2) testing of new stent material and new designs for these biliary stents; and 3) the recent introduction of self-expandable metal stents. Each method has its own merits as well as specific problems. This article reviews the pathogenesis of biofilm formation on the biliary stents and the latest status of research in avoiding the problem of stent occlusion.

Background: Portal hypertensive gastropathy is characterized by a hyperdynamic circulatory state in the gastric mucosa. We assessed the effect of bolus injection of somatostatin on gastric mucosal perfusion in a rat model of portal hypertension. We tested the hypothesis that somatostatin will reduce gastric mucosal perfusion in portal hypertension.Methods: Portal hypertension was induced by two-stage portal vein ligation (PVL). Two weeks after PVL significant elevation of portal venous pressure was demonstrated. Gastric mucosal hernodynamic changes were measured by reflectance spectrophotometry, which records the indexes of mucosal hemoglobin oxygen saturation (ISO2) and mucosal hemoglobin concentration (IHB).Results: After an intravenous bolus of 1 pg somatostatin significant reductions of ISO2 and IHB were demonstrated in the rats with PVL (ISO2, -34 ± 5%; IHB, -15 ± 2%) and the controls (ISO2, -26 ± 4%; IHB, -15 ± 2%). A dose-response relationship was shown by using 0.01, 0.1, and 1 μg of somatostatin. Somatostatin did not induce other hemodynamic changes except a transient drop in systemic blood pressure of ISO2.Conclusion: The significant reductions of gastric mucosal ISO2 and IHB by somatostatin support the hypothesis that somatostatin is capable of attenuating the hyperdynamic circulatory state in the gastric mucosa of rats with portal hypertension and may have a beneficial effect on portal hypertensive gastropathy. This hypothesis deserves to be evaluated in clinical studies.

There has been controversy regarding the relationship between Helicobacter pyloriand perforated peptic ulcer, which is known to have a high recurrence rate if only simple patch repair is performed. The aim of this study was to evaluate the association between H. pyloriinfection and intake of non-steroidal anti-inflammatory drugs (NSAIDs) in patients with perforated duodenal ulcers. Of the 73 patients recruited over a 16-month period, 51 (70 per cent) had evidence of H. pyloriinfection by intra-operative gastroscopy and antral biopsies. The infection rate rose to 80 per cent if NSAID users were excluded. The H. pylori-infected group was significantly younger (mean 47.6 versus62.5 years), with a male preponderance (49 of 51 versus14 of 22 patients), and had significantly less NSAID consumption (three of 51 versusten of 22) and more prolonged dyspepsia (40 of 51 versusten of 22), compared with H. pylori-negative patients. H. pyloriinfection probably plays an important role in the causation of non-NSAID-induced duodenal ulcer perforation. Whether eradication of the bacteria can alleviate the strong ulcer diathesis in this subgroup of patients is unknown.