Search

Arteriosclerosis is intimately associated with cardiovascular diseases. Recently, evidence accumulated that infection with Helicobacter pylori cagA-positive strains, which causes gastritis, peptic ulceration, and gastric cancer, is also involved in the development of arteriosclerosis. The cagA-encoded CagA protein is injected into the attached gastric epithelial cells via the type IV secretion system. We previously showed that CagA-containing exosomes are secreted from CagA-injected gastric epithelial cells and enter the systemic blood circulation, delivering CagA into endothelial cells. In the present study, transgenic mice were established in which CagA was selectively expressed in endothelial cells by Cre-loxP system. Treatment of the mice with a high-fat diet revealed that atherogenic lesions were induced in mice expressing CagA in vascular endothelial cells but not in CagA-nonexpressing mice. To investigate the effects of CagA on endothelial cells, we also established conditional CagA-expressing human vascular endothelial cells using the Tet-on system. Upon induction of CagA, a dramatic change in cell morphology was observed that was concomitantly associated with the loss of the endothelial cells to form tube-like structures. Induction of CagA also activated the pro-inflammatory transcription factor STAT3. Thus, exosome-delivered CagA deregulates signals that activates STAT3 in endothelial cells, which accelerates inflammation that promotes arteriosclerosis/atherosclerosis.

Infection with cagA-positive Helicobacter pylori strains plays an etiological role in the development of gastric cancer. The CagA protein is injected into gastric epithelial cells through a bacterial Type IV secretion system. Inside the host cells, CagA promiscuously associates with multiple host cell proteins including the prooncogenic phosphatase SHP2 that is required for full activation of the Ras-ERK pathway. CagA-SHP2 interaction aberrantly activates SHP2 and thereby deregulates Ras-ERK signaling. Cancer is regarded as a disease of the genome, indicating that H. pylori-mediated gastric carcinogenesis is also associated with genomic alterations in the host cell. Indeed, accumulating evidence has indicated that H. pylori infection provokes DNA double-stranded breaks (DSBs) by both CagA-dependent and CagA-independent mechanisms. DSBs are repaired by either error-free homologous recombination (HR) or error-prone non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ). Infection with cagA-positive H. pylori inhibits RAD51 expression while dampening cytoplasmic-to-nuclear translocalization of BRCA1, causing replication fork instability and HR defects (known as "BRCAness"), which collectively provoke genomic hypermutation via non-HR-mediated DSB repair. H. pylori also subverts multiple DNA damage responses including DNA repair systems. Infection with H. pylori additionally inhibits the function of the p53 tumor suppressor, thereby dampening DNA damage-induced apoptosis, while promoting proliferation of CagA-delivered cells. Therefore, H. pylori cagA-positive strains promote abnormal expansion of cells with BRCAness, which dramatically increases the chance of generating driver gene mutations in the host cells. Once such driver mutations are acquired, H. pylori CagA is no longer required for subsequent gastric carcinogenesis (Hit-and-Run carcinogenesis).

PAR1b is a cytoplasmic serine/threonine kinase that controls cell polarity and cell–cell interaction by regulating microtubule stability while mediating cytoplasmic-to-nuclear translocation of BRCA1. PAR1b is also a cellular target of the CagA protein of Helicobacter pylori, which leads to chronic infection causatively associated with the development of gastric cancer. The CagA-PAR1b interaction inactivates the kinase activity of PAR1b and thereby dampens PAR1b-mediated BRCA1 phosphorylation, which reduces the level of nuclear BRCA1 and thereby leads to BRCAness and BRCAness-associated genome instability underlying gastric carcinogenesis. While PAR1b can multimerize within the cells, little is known about the mechanism and functional role of PAR1b multimerization. We found in the present study that PAR1b was multimerized in vitro by binding with nucleic acids (both single- and double-stranded DNA/RNA) via the spacer region in a manner independent of nucleic-acid sequences, which markedly potentiated the kinase activity of PAR1b. Consistent with these in vitro observations, cytoplasmic introduction of double-stranded DNA or expression of single-stranded RNA increased the PAR1b kinase activity in the cells. These findings indicate that the cytoplasmic DNA/RNA contribute to nuclear accumulation of BRCA1 by constitutively activating/potentiating cytoplasmic PAR1b kinase activity, which is subverted in gastric epithelial cells upon delivery of H. pylori CagA oncoprotein.

In addition to acting as a transcriptional co-activator, YAP1 directly mediates translocalization of the pro-oncogenic phosphatase SHP2 from the cytoplasm to nucleus. In the cytoplasm, SHP2 potentiates RAS–ERK signaling, which promotes cell proliferation and cell motility, whereas in the nucleus, it mediates gene regulation. As a result, elucidating the details of SHP2 trafficking is important for understanding its biological roles, including in cancer. YAP1 comprises multiple splicing isoforms defined in part by the presence (as in YAP1-2γ) or absence (as in YAP1-2α) of a γ-segment encoded by exon 6 that disrupts a critical leucine zipper. Although the disruptive segment is known to reduce co-activator function, it is unclear how this element impacts the physical and functional relationships between YAP1 and SHP2. To explore this question, we first demonstrated that YAP1-2γ cannot bind SHP2. Nevertheless, YAP1-2γ exhibits stronger mitogenic and motogenic activities than does YAP1-2α because the YAP1-2α–mediated delivery of SHP2 to the nucleus weakens cytoplasmic RAS–ERK signaling. However, YAP1-2γ confers less in vivo tumorigenicity than does YA1-2α by recruiting tumor-inhibitory macrophages. Mechanistically, YAP1-2γ transactivates and the YAP1-2α–SHP2 complex transrepresses the monocyte/macrophage chemoattractant CCL2. Thus, cell-intrinsic and cell-extrinsic pro-oncogenic YAP1 activities are inversely regulated by alternative splicing of exon 6. Notably, oncogenic KRAS down-regulates the SRSF3 splicing factor that prevents exon 6 skipping, thereby creating a YAP1-2α–dominant situation that supports a “cold” immune microenvironment.

Natural disasters like earthquakes and heavy rainfall events induce slope failures and other disasters. Proper determination of the water retention properties of porous mediums is necessary. The conventional Soil Water Characteristics Curve (SWCC) determination techniques are limited due to their complexity and lengthy time required. A highly accurate simple remolded and undisturbed samples SWCC determination method that requires a short time is lacking. A novel automatic system which adopts the continuous pressurization method and allows the direct determination of the SWCC in a short time suitable for use with remolded and undisturbed samples was developed. The accuracy of the newly developed system was validated, and it was shown to have excellent repeatability. It is a reliable and direct method that requires a short time for the SWCC determination, with the drying and wetting SWCCs obtainable in less than 10% of the time required using the conventional multi-step flow method. Consideration of the pore water pressure was shown to be necessary when calculating the matric suction utilizing the axis-translation technique. Finally, because remolded samples do not properly represent the in-situ natural conditions, the significant discrepancies should be carefully considered when conducting analysis and proposing countermeasures against unsaturated soil-related Geo-disasters.

Chronic infection with Helicobacter pylori cagA‐positive strains is causally associated with the development of gastric diseases, most notably gastric cancer. The cagA‐encoded CagA protein, which is injected into gastric epithelial cells by bacterial type IV secretion, undergoes tyrosine phosphorylation at the Glu‐Pro‐Ile‐Tyr‐Ala (EPIYA) segments (EPIYA‐A, EPIYA‐B, EPIYA‐C, and EPIYA‐D), which are present in various numbers and combinations in its C‐terminal polymorphic region, thereby enabling CagA to promiscuously interact with SH2 domain‐containing host cell proteins, including the prooncogenic SH2 domain‐containing protein tyrosine phosphatase 2 (SHP2). Perturbation of host protein functions by aberrant complex formation with CagA has been considered to contribute to the development of gastric cancer. Here we show that SHIP2, an SH2 domain‐containing phosphatidylinositol 5′‐phosphatase, is a hitherto undiscovered CagA‐binding host protein. Similar to SHP2, SHIP2 binds to the Western CagA‐specific EPIYA‐C segment or East Asian CagA‐specific EPIYA‐D segment through the SH2 domain in a tyrosine phosphorylation‐dependent manner. In contrast to the case of SHP2, however, SHIP2 binds more strongly to EPIYA‐C than to EPIYA‐D. Interaction with CagA tethers SHIP2 to the plasma membrane, where it mediates production of phosphatidylinositol 3,4‐diphosphate [PI(3,4)P2]. The CagA‐SHIP2 interaction also potentiates the morphogenetic activity of CagA, which is caused by CagA‐deregulated SHP2. This study indicates that initially delivered CagA interacts with SHIP2 and thereby strengthens H. pylori‐host cell attachment by altering membrane phosphatidylinositol compositions, which potentiates subsequent delivery of CagA that binds to and thereby deregulates the prooncogenic phosphatase SHP2., The SH2 domain‐containing phosphatidylinositol 5′‐phosphatase, SHIP2, is a hitherto undiscovered CagA‐binding host protein. The CAgA‐SHIP2 interaction potentiates Helicobacter pylori‐mediated CagA delivery into gastric epithelial cells, which then promotes the formation of the oncogenic CagA‐SHP2 complex.

The initial step in bacterial infection is adherence of the bacterium to the target cell surface.

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen

Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor for gastric cancer. The cagA gene product, CagA, is delivered into gastric epithelial cells via the bacterial type IV secretion system. Delivered CagA then undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in its C-terminal region and acts as an oncogenic scaffold protein that physically interacts with multiple host signaling proteins in both tyrosine phosphorylation-dependent and -independent manners. Analysis of CagA using in vitro cultured gastric epithelial cells has indicated that the nonphysiological scaffolding actions of CagA cell-autonomously promote the malignant transformation of the cells by endowing the cells with multiple phenotypic cancer hallmarks: sustained proliferation, evasion of growth suppressors, invasiveness, resistance to cell death, and genomic instability. Transgenic expression of CagA in mice leads to in vivo oncogenic action of CagA without any overt inflammation. The in vivo oncogenic activity of CagA is further potentiated in the presence of chronic inflammation. Since Helicobacter pylori infection triggers a proinflammatory response in host cells, a feedforward stimulation loop that augments the oncogenic actions of CagA and inflammation is created in CagA-injected gastric mucosa. Given that Helicobacter pylori is no longer colonized in established gastric cancer lesions, the multistep nature of gastric cancer development should include a “hit-and-run” process of CagA action. Thus, acquisition of genetic and epigenetic alterations that compensate for CagA-directed cancer hallmarks may be required for completion of the “hit-and-run” process of gastric carcinogenesis.

Since its discovery, Helicobacter pylori has been identified as the causative agent of various gastric diseases. H. pylori produces myriads of disease-associated virulence factors. These bacterial determinants can be distinguished as cell-binding factors, immunoregulatory components, survival factors, toxins, and effector proteins. For most of these factors there is consensus about their classification. However, there is a strong dispute in the literature as to whether one of the best-studied factors, CagA, represents a toxin or not. CagA displays unique functions that are clearly different from conventional toxins, and CagA counteracts the activities of an established H. pylori toxin, VacA. Canonical toxins commonly have specific (and narrow) targets, can act even in the absence of the bacterial cell, and elicit acute damage to host cells. However, there is still no agreement on the classification of CagA. Here we discuss whether CagA acts as a toxin, and propose a classification consensus for CagA.

In most cases of sporadic colorectal cancers, tumorigenesis is a multistep process, involving genomic alterations in parallel with morphologic changes. In addition, accumulating evidence suggests that the human gut microbiome is linked to the development of colorectal cancer. Here we performed fecal metagenomic and metabolomic studies on samples from a large cohort of 616 participants who underwent colonoscopy to assess taxonomic and functional characteristics of gut microbiota and metabolites. Microbiome and metabolome shifts were apparent in cases of multiple polypoid adenomas and intramucosal carcinomas, in addition to more advanced lesions. We found two distinct patterns of microbiome elevations. First, the relative abundance of Fusobacterium nucleatum spp. was significantly (P

Summary This protocol visualizes dynamic interaction between a transmembrane protein and an intracellular protein induced by clusterization/oligomerization of the transmembrane protein. Association-dissociation of the intracellular region of the transmembrane protein with cytoplasmic protein(s) is detected by proximity ligation assay. Since a transmembrane protein often resists extraction, biochemical analysis of its dynamic interaction with cytoplasmic effectors is cumbersome. This protocol quantitatively visualizes protein-protein interaction occurring in the membrane periphery, providing a powerful tool to elucidate signal transduction across the membrane. For complete details on the use and execution of this protocol, please refer to Ooki et al. (2019)., Graphical Abstract, Highlights • Antibody-mediated cross-linking induces functional clustering of membrane protein • PLA can visualize extracellular-signal-dependent intracellular protein interaction • This protocol is applicable for the analysis of dynamic protein network formation, This protocol visualizes dynamic interaction between a transmembrane protein and an intracellular protein induced by clusterization/oligomerization of the transmembrane protein. Association-dissociation of the intracellular region of the transmembrane protein with cytoplasmic protein(s) is detected by proximity ligation assay. Since a transmembrane protein often resists extraction, biochemical analysis of its dynamic interaction with cytoplasmic effectors is cumbersome. This protocol quantitatively visualizes protein-protein interaction occurring in the membrane periphery, providing a powerful tool to elucidate signal transduction across the membrane.

Chronic infection with Helicobacter pylori, especially cagA-positive strains, plays an etiological role in the development of gastric cancer. The Cytotoxin-associated gene A (CagA) protein, encoded by cagA, is delivered into gastric epithelial cells via bacterial type IV secretion, where it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Host cell-delivered CagA then acts as a nonphysiological scaffold/hub by interacting with multiple signaling molecules, most notably the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1b, in both tyrosine phosphorylation-dependent and -independent manners. CagA-mediated manipulation of intracellular signaling promotes neoplastic transformation of gastric epithelial cells. Indeed, transgenic expression of CagA in experimental animals has confirmed the oncogenic potential of the bacterial protein. Structural polymorphism of CagA influences its scaffold function, which may underlie the geographic difference in the incidence of gastric cancer. Meanwhile, since CagA is no longer required for the maintenance of established gastric cancer, CagA appears to promote gastric carcinogenesis through a hit-and-run mechanism. In this chapter, these facets of CagA and their impact on gastric carcinogenesis will be discussed in detail.

Background & Aims: CD44 variant 9 (CD44v9)-positive cancer stem-like cells strongly contribute to the development and recurrence of gastric cancer. However, the origin of CD44v9-positive cells is uncertain. Methods: CD44v9, β-catenin, and epithelial splicing regulatory protein 1 signals were assessed by real-time reverse-transcription polymerase chain reaction, immunoblot analysis, or immunofluorescence microscopy. Capping actin protein of muscle Z-line α subunit 1 (CAPZA1) expression was assessed by immunoblot analysis or immunohistochemical analysis of Mongolian gerbils' gastric mucosa or human biopsy specimens. Levels of oxidative stress were assessed by measuring malondialdehyde and protein carbonylation. Histone H3 acetylation levels in the CAPZA1 proximal promoter region were measured by using chromatin immunoprecipitation analysis with an antibody against the acetylated histone H3 in human gastric carcinoma cell line (AGS) cells. Results: CD44v9 is expressed in CAPZA1-overexpressing cells in human gastric cancer tissues. CAPZA1 overexpression enhanced expression of β-catenin, which is a transcription factor for CD44, and epithelial splicing regulatory protein 1, which increases alternative splicing of CD44 to generate CD44v9. CAPZA1-overexpressing cells after cytotoxin-associated gene A accumulation showed CD44v9 expression by inducing nuclear accumulation of β-catenin, concomitant with the enhancement of expression of Sal-like protein 4 and Krüppel-like factor 5, which encode reprogramming factors. Oxidative stress increased the CAPZA1 expression in AGS cells through the enhancement of histone H3 acetylation of CAPZA1 promoter. CAPZA1 expression was increased depending on oxidative stress in H pylori–infected gastric mucosa. Conclusions: CD44v9 expression is evoked from CAPZA1-overexpressing cells after accumulation of cytotoxin-associated gene A. Our findings provide important insights into the mechanisms underlying the development of CD44v9-positive cells. Keywords: Autophagy, CagA, Cancer Stem Cells, CD44v9, Eradication Therapy

The initial step in bacterial infection is adherence of the bacterium to the target cell surface. Helicobacter pylori exploits the interaction of bacterial adhesin protein HopQ with human epithelial CEACAMs (CEACAM1, 5, and 6) to stably adhere to gastric epithelial cells, which is necessary for delivery of the H. pylori CagA oncoprotein into the epithelial cells via a type IV secretion system. In contrast to human CEACAMs, however, HopQ does not interact with Ceacam1 (mouse CEACAM1) in vitro or in CHO cells ectopically expressing Ceacam1. Since the mouse genome lacks Ceacam5 and Ceacam6, no significant HopQ–Ceacam interaction may occur in mouse gastric epithelial cells. Here, we found that the mouse stomach has a much lower expression level of Ceacam1 than the expression level of CEACAM1 in the human stomach. Consistently, mouse gastric epithelial cells resist CagA delivery by cagA-positive H. pylori, and the delivery is restored by ectopic expression of human CEACAM1 or CEACAM5 in mouse gastric epithelial cells. Thus, despite the fact that mice are routinely used for H. pylori infection studies, a low expression level of Ceacam1 in the mouse stomach together with the loss or greatly reduced interaction of HopQ with Ceacams make the mouse an inappropriate model for studying the role of H. pylori-delivered CagA in gastric pathogenesis, including the development of gastric cancer.

The proteins from the Fanconi Anemia (FA) pathway of DNA repair maintain DNA replication fork integrity by preventing the unscheduled degradation of nascent DNA at regions of stalled replication forks. Here, we ask if the bacterial pathogen H. pylori exploits the fork stabilisation machinery to generate double stand breaks (DSBs) and genomic instability. Specifically, we study if the H. pylori virulence factor CagA generates host genomic DSBs through replication fork destabilisation and collapse. An inducible gastric cancer model was used to examine global CagA-dependent transcriptomic and proteomic alterations, using RNA sequencing and SILAC-based mass spectrometry, respectively. The transcriptional alterations were confirmed in gastric cancer cell lines infected with H. pylori. Functional analysis was performed using chromatin fractionation, pulsed-field gel electrophoresis (PFGE), and single molecule DNA replication/repair fiber assays. We found a core set of 31 DNA repair factors including the FA genes FANCI, FANCD2, BRCA1, and BRCA2 that were downregulated following CagA expression. H. pylori infection of gastric cancer cell lines showed downregulation of the aforementioned FA genes in a CagA-dependent manner. Consistent with FA pathway downregulation, chromatin purification studies revealed impaired levels of Rad51 but higher recruitment of the nuclease MRE11 on the chromatin of CagA-expressing cells, suggesting impaired fork protection. In line with the above data, fibre assays revealed higher fork degradation, lower fork speed, daughter strands gap accumulation, and impaired re-start of replication forks in the presence of CagA, indicating compromised genome stability. By downregulating the expression of key DNA repair genes such as FANCI, FANCD2, BRCA1, and BRCA2, H. pylori CagA compromises host replication fork stability and induces DNA DSBs through fork collapse. These data unveil an intriguing example of a bacterial virulence factor that induces genomic instability by interfering with the host replication fork stabilisation machinery.

High-molecular-weight hyaluronan acts as a ligand of the tumor-suppressive Hippo signal, whereas degradation of hyaluronan from a high-molecular-weight form to a low-molecular-weight forms by hyaluronidase 2 inhibits Hippo signal activation and thereby activates the pro-oncogenic transcriptional coactivator yes-associated protein (YAP), which creates a cancer-predisposing microenvironment and drives neoplastic transformation of cells through both cell-autonomous and non-cell-autonomous mechanisms. In fact, accumulation of low-molecular-weight hyaluronan in tissue stroma is observed in many types of cancers. Since inhibition of YAP activity suppresses tumor growth in vivo, pharmacological intervention of the Hippo-YAP signal is an attractive approach for future drug development. In this review, pharmacological intervention of excessive hyaluronan degradation as a novel approach for inhibition of the Hippo-YAP signal is also discussed. Development of hyaluronidase inhibitors may provide novel therapeutic strategies for human malignant tumors.

The Great East Japan Earthquake and caused the tsunami on March 11th, 2011 that destroyed much of the fisheries infrastructure along the North Pacific coast of Japan. After this disaster, most of the oyster growers of Karakuwa district have been sitting in a shocked condition in the evacuation facility. However, the role of restoration support by iLINK Inc. who conveyed the earnest desire of consumers for Karakuwa oysters and the aid from home and abroad awakened the grower’s mind back to the ocean. To restart oyster farming, it was necessary to overcome difficulties arisen before the tsunami disaster. Through the continued discussion, members of Karakuwa Branch, Miyagi Fisheries Cooperative Association decided new agreement to solve problems such as overcrowded raft setting, irregular growth of oyster caused by interference of the current flow and old customs blocking efficient utilization of fishing ground. Based on these agreements an improved and rational oyster farming system has started with a goal of rationalizing production for the future. The interactions with the restoration supporters have enabled the fishermen to look at their work from a new prospective. Now the oyster growers have realized the significance of oyster farming which is watched with keen interest by the consumers. Interchanges between restoration supporters for tsunami-affected oyster farming inspired the minds of growers’ and contributed to the innovative and rational oyster farming.

Helicobacter pylori is the first bacterium formally recognized to play a causative role in human malignancies, gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Evidence accumulates that H. pylori cagA-positive strains play a crucial role in the neoplastic transformation of mammalian cells. The cagA-encoded CagA protein is delivered into the host cells via bacterial type IV secretion, where it interacts with and thereby aberrantly activates pro-oncogenic phosphatase SHP2. The CagA-SHP2 interaction requires tyrosine phosphorylation of CagA at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. The incidences of gastric cancer in East Asian countries such as Japan, China, and Korea are among the highest worldwide. A vast majority of H. pylori circulating in East Asia produce a CagA variant termed East Asian CagA, which possesses the SHP2-binding EPIYA motif (EPIYA-D) that is substantially diverged in sequence from the SHP2-binding EPIYA motif (EPIYA-C) of CagA isolated in the rest of the world (Western CagA). Tyrosine-phosphorylated EPIYA-D interacts with SHP2 approximately two orders of magnitude stronger than tyrosine-phosphorylated EPIYA-C does. The strong SHP2 binding of East Asian CagA is achieved by a cryptic interaction between the phenylalanine residue located at the +5 position from the phospho-tyrosine in EPIYA-D and a small hollow on the N-SH2 phosphopeptide-binding floor, the latter of which cannot be created by the corresponding aspartic acid in EPIYA-C. Thus, a variation in a single amino-acid residue determines the magnitude for the pathogenic/oncogenic action of CagA, which may influence the worldwide landscape in the incidence of H. pylori-associated malignancies, especially gastric cancer.

Summary Infection with CagA-producing Helicobacter pylori plays a causative role in the development of gastric cancer. Upon delivery into gastric epithelial cells, CagA deregulates prooncogenic phosphatase SHP2 while inhibiting polarity-regulating kinase PAR1b through complex formation. Here, we show that CagA/PAR1b interaction subverts nuclear translocation of BRCA1 by inhibiting PAR1b-mediated BRCA1 phosphorylation. It hereby induces BRCAness that promotes DNA double-strand breaks (DSBs) while disabling error-free homologous recombination-mediated DNA repair. The CagA/PAR1b interaction also stimulates Hippo signaling that circumvents apoptosis of DNA-damaged cells, giving cells time to repair DSBs through error-prone mechanisms. The DSB-activated p53-p21Cip1 axis inhibits proliferation of CagA-delivered cells, but the inhibition can be overcome by p53 inactivation. Indeed, sequential pulses of CagA in TP53-mutant cells drove somatic mutation with BRCAness-associated genetic signatures. Expansion of CagA-delivered cells with BRCAness-mediated genome instability, from which CagA-independent cancer-predisposing cells arise, provides a plausible “hit-and-run mechanism” of H. pylori CagA for gastric carcinogenesis.

Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor of gastric cancer. The cagA gene-encoded CagA protein is delivered into gastric epithelial cells via bacterial type IV secretion, where it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Delivered CagA then acts as a non-physiological scaffold/hub protein by interacting with multiple host signaling molecules, most notably the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1/MARK, in both tyrosine phosphorylation-dependent and -independent manners. CagA-mediated manipulation of intracellular signaling promotes neoplastic transformation of gastric epithelial cells. Transgenic expression of CagA in experimental animals has confirmed the oncogenic potential of the bacterial protein. Structural polymorphism of CagA influences its scaffold function, which may underlie the geographic difference in the incidence of gastric cancer. Since CagA is no longer required for the maintenance of established gastric cancer cells, studying the role of CagA during neoplastic transformation will provide an excellent opportunity to understand molecular processes underlying "Hit-and-Run" carcinogenesis.

// Rony K. Roy 1, * , Michal M. Hoppe 1, * , Supriya Srivastava 1 , Animesh Samanta 2 , Neel Sharma 3 , Kar Tong Tan 1 , Henry Yang 1 , Dominic C. Voon 1 , Brendan Pang 4 , Ming Teh 4 , Naoko Murata-Kamiya 5 , Masanori Hatakeyama 5 , Young-Tae Chang 2 , Wei Peng Yong 1, 6 , Yoshiaki Ito 1, 6 , Khek Yu Ho 2, 6 , Patrick Tan 1, 6 , Richie Soong 1 , Phillip H. Koeffler 1, 2 , Khay Guan Yeoh 2, 6 , Anand D. Jeyasekharan 1, 6 1 Cancer Science Institute of Singapore, National University of Singapore, Singapore 2 Department of Chemistry, National University of Singapore, Singapore 3 Division of Gastroenterology and Hepatology, National University Hospital, Singapore 4 Department of Pathology, National University Hospital, Singapore 5 University of Tokyo, Tokyo, Japan 6 Singapore Gastric Cancer Consortium, National University of Singapore, Singapore * These authors have contributed equally to this work Correspondence to: Khay Guan Yeoh, email: mdcykg@nus.edu.sg Anand D. Jeyasekharan, email: csiadj@nus.edu.sg Keywords: gastric cancer, biomarker, Helicobacter pylori, CEACAM6, endoscopy Received: February 19, 2016 Accepted: May 23, 2016 Published: July 11, 2016 ABSTRACT Early detection of gastric cancers saves lives, but remains a diagnostic challenge. In this study, we aimed to identify cell-surface biomarkers of early gastric cancer. We hypothesized that a subset of plasma membrane proteins induced by the Helicobacter pylori oncoprotein CagA will be retained in early gastric cancers through non-oncogene addiction. An inducible system for expression of CagA was used to identify differentially upregulated membrane protein transcripts in vitro . The top hits were then analyzed in gene expression datasets comparing transcriptome of gastric cancer with normal tissue, to focus on markers retained in cancer. Among the transcripts enriched upon CagA induction in vitro , a significant elevation of CEACAM6 was noted in gene expression datasets of gastric cancer. We used quantitative digital immunohistochemistry to measure CEACAM6 protein levels in tissue microarrays of gastric cancer. We demonstrate an increase in CEACAM6 in early gastric cancers, when compared to matched normal tissue, with an AUC of 0.83 for diagnostic validity. Finally, we show that a fluorescently conjugated CEACAM6 antibody binds avidly to freshly resected gastric cancer xenograft samples and can be detected by endoscopy in real time. Together, these results suggest that CEACAM6 upregulation is a cell surface response to H. pylori CagA, and is retained in early gastric cancers. They highlight a novel link between CEACAM6 expression and CagA in gastric cancer, and suggest CEACAM6 to be a promising biomarker to aid with the fluorescent endoscopic diagnosis of early neoplastic lesions in the stomach.

Pragmin is one of the few mammalian proteins containing the Glu-Pro-Ile-Tyr-Ala (EPIYA) tyrosine-phosphorylation motif that was originally discovered in the Helicobacter pylori CagA oncoprotein. Following delivery into gastric epithelial cells by type IV secretion and subsequent tyrosine phosphorylation at the EPIYA motifs, CagA serves as an oncogenic scaffold/adaptor that promiscuously interacts with SH2 domain-containing mammalian proteins such as the Src homology 2 (SH2) domain-containing protein tyrosine phosphatase-2 (SHP2) and the C-terminal Src kinase (Csk), a negative regulator of Src family kinases. Like CagA, Pragmin also forms a physical complex with Csk. In the present study, we found that Pragmin directly binds to Csk by the tyrosine-phosphorylated EPIYA motif. The complex formation potentiates kinase activity of Csk, which in turn phosphorylates Pragmin on tyrosine-238 (Y238), Y343, and Y391. As Y391 of Pragmin comprises the EPIYA motif, Pragmin-Csk interaction creates a feed-forward regulatory loop of Csk activation. Together with the finding that Pragmin and Csk are colocalized to focal adhesions, these observations indicate that the Pragmin-Csk interaction, triggered by Pragmin EPIYA phosphorylation, robustly stimulates the kinase activity of Csk at focal adhesions, which direct cell-matrix adhesion that regulates cell morphology and cell motility. As a consequence, expression of Pragmin and/or Csk in epithelial cells induces an elongated cell shape with elevated cell scattering in a manner that is mutually dependent on Pragmin and Csk. Deregulation of the Pragmin-Csk axis may therefore induce aberrant cell migration that contributes to tumor invasion and metastasis.

Bacterial infections typically elicit a strong Heat Shock Response (HSR) in host cells. However, the gastric pathogen Helicobacter pylori has the unique ability to repress this response, the mechanism of which has yet to be elucidated. This study sought to characterize the underlying mechanisms by which H. pylori down-modulates host HSP expression upon infection. Examination of isogenic mutant strains of H. pylori defective in components of the type IV secretion system (T4SS), identified the secretion substrate, CagA, to be essential for down-modulation of the HSPs HSPH1 (HSP105), HSPA1A (HSP72), and HSPD1 (HSP60) upon infection of the AGS gastric adenocarcinoma cell line. Ectopic expression of CagA by transient transfection was insufficient to repress HSP expression in AGS or HEK293T cells, suggesting that additional H. pylori factors are required for HSP repression. RT-qPCR analysis of HSP gene expression in AGS cells infected with wild-type H. pylori or isogenic cagA-deletion mutant found no significant change to account for reduced HSP levels. In summary, this study identified CagA to be an essential bacterial factor for H. pylori-mediated suppression of host HSP expression. The novel finding that HSPH1 is down-modulated by H. pylori further highlights the unique ability of H. pylori to repress the HSR within host cells. Elucidation of the mechanism by which H. pylori achieves HSP repression may prove to be beneficial in the identification of novel mechanisms to inhibit the HSR pathway and provide further insight into the interactions between H. pylori and the host gastric epithelium.

Current X-ray sources—such as high-flux synchrotron radiation and XFEL—enable us to determine the tertiary structures of proteins at medium or low resolution even from crystals with poor quality. However, high-resolution crystal structures are still required to obtain detailed information about protein structures in the pharmaceutical and biochemical sciences. To improve the crystal quality, several types of post-crystallization treatments, including soaking experiments, have been utilized. However, such treatments do not always improve the crystal quality, and a new method of crystal-quality enhancement is eagerly awaited. Here, we propose a novel strategy for the post-crystallization treatment, the multistep soaking method, in which a crystal is sequentially soaked in 2–3 different cryoprotectant solutions. The sequential use of different cryoprotectants significantly expands the number of options for subsequent post-crystallization treatment. Using the new method, we successfully improved the crystal q...

The water retention test involving the stepwise pressurization method (SPM) and a pressure plate extractor has been mainly used to obtain the soil–water characteristic curve (SWCC) of soils. The disadvantage of this test is that the measurement of the drying and wetting paths of the SWCC is time consuming. Thus, a water retention test involving a continuous pressurization method (CPM) in this paper was proposed, and the experiments for three kinds of soil samples were performed. Unlike the SPM, the pore air pressure in the CPM is continuously given to the specimen at a constant pressurization rate. In particular, for the measurement of the transient pore water pressure inside specimen according to the applied pore air pressure, a microtensiometer (height: 25 mm, diameter: 3 mm) was placed vertically at the center of the specimen. The suction for the specimen was defined as the difference between the applied pore air pressure and the measured pore water pressure. It was confirmed that the testing time for the drying and wetting paths of the SWCC in the test involving the CPM was remarkably shortened. The results of the CPM for its verification were compared with those of the SPM under the same initial condition of the specimen. The two test results showed good agreement regardless of the soil samples. Furthermore, the effect of the pressurization rate of the pore air pressure for the CPM was examined. Finally, the proposed water retention test involving the CPM was found to be highly efficient and accurately measure the drying and wetting paths of the SWCC in a short time.

Through this paper, a sampling methodology and a novel full automatic system adopting the continuous pressurization method which is capable of determining the Soil Water Characteristics Curve (SWCC) for both remoulded and undisturbed samples in a very short time were developed. The proposed system was validated by comparing the SWCCs of standard testing soils obtained using the developed system to the SWCCs obtained using a conventional method. Remoulded and undisturbed natural soil samples were tested, where the degree of disturbance influence on the obtained SWCC was discussed. In addition, the undisturbed samples containing moulds material influence on the obtained SWCC was investigated. It was found that remoulded samples do not properly represent the in-situ conditions with significant error that should be carefully considered when conducting analysis and proposing countermeasures against unsaturated soils related Geo-disasters. In addition, the material which the containing mould is made from has minor influence on the obtained SWCC which can be neglected. Finally, it can be concluded that the developed undisturbed soil water characteristics curve obtaining system is direct, rapid, reliable and simple. In addition, the proposed undisturbed sampling and testing methodology can be used to accurately evaluate the spatial variations of the SWCC regardless the heterogeneity of the soil profile.

Helicobacter pylori is the first bacterium formally recognized to play a causative role in human malignancies, gastric cancer and gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Evidence accumulates that H. pylori cagA-positive strains play a crucial role in the neoplastic transformation of mammalian cells. The cagA-encoded CagA protein is delivered into the host cells via bacterial type IV secretion, where it interacts with and thereby aberrantly activates pro-oncogenic phosphatase SHP2. The CagA-SHP2 interaction requires tyrosine phosphorylation of CagA at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. The incidences of gastric cancer in East Asian countries such as Japan, China, and Korea are among the highest worldwide. A vast majority of H. pylori circulating in East Asia produce a CagA variant termed East Asian CagA, which possesses the SHP2-binding EPIYA motif (EPIYA-D) that is substantially diverged in sequence from the SHP2-binding EPIYA motif (EPIYA-C) of CagA isolated in the rest of the world (Western CagA). Tyrosine-phosphorylated EPIYA-D interacts with SHP2 approximately two orders of magnitude stronger than tyrosine-phosphorylated EPIYA-C does. The strong SHP2 binding of East Asian CagA is achieved by a cryptic interaction between the phenylalanine residue located at the +5 position from the phospho-tyrosine in EPIYA-D and a small hollow on the N-SH2 phosphopeptide-binding floor, the latter of which cannot be created by the corresponding aspartic acid in EPIYA-C. Thus, a variation in a single amino-acid residue determines the magnitude for the pathogenic/oncogenic action of CagA, which may influence the worldwide landscape in the incidence of H. pylori-associated malignancies, especially gastric cancer.

In most cases of sporadic colorectal cancers, tumorigenesis is a multistep process, involving genomic alterations in parallel with morphologic changes. In addition, accumulating evidence suggests that the human gut microbiome is linked to the development of colorectal cancer. Here we performed fecal metagenomic and metabolomic studies on samples from a large cohort of 616 participants who underwent colonoscopy to assess taxonomic and functional characteristics of gut microbiota and metabolites. Microbiome and metabolome shifts were apparent in cases of multiple polypoid adenomas and intramucosal carcinomas, in addition to more advanced lesions. We found two distinct patterns of microbiome elevations. First, the relative abundance of Fusobacterium nucleatum spp. was significantly (P 0.005) elevated continuously from intramucosal carcinoma to more advanced stages. Second, Atopobium parvulum and Actinomyces odontolyticus, which co-occurred in intramucosal carcinomas, were significantly (P 0.005) increased only in multiple polypoid adenomas and/or intramucosal carcinomas. Metabolome analyses showed that branched-chain amino acids and phenylalanine were significantly (P 0.005) increased in intramucosal carcinomas and bile acids, including deoxycholate, were significantly (P 0.005) elevated in multiple polypoid adenomas and/or intramucosal carcinomas. We identified metagenomic and metabolomic markers to discriminate cases of intramucosal carcinoma from the healthy controls. Our large-cohort multi-omics data indicate that shifts in the microbiome and metabolome occur from the very early stages of the development of colorectal cancer, which is of possible etiological and diagnostic importance.

Summary High-molecular-weight hyaluronan, a major component of the extracellular matrix, is anti-oncogenic, whereas low-molecular-weight hyaluronan is pro-oncogenic, though the mechanisms underlying the size-dependent opposite bioactivities of hyaluronan remain uncertain. We show here that treatment with high-molecular-weight hyaluronan stimulates tumor-suppressive Hippo signaling in breast epithelial cells. Mechanistically, clustering of the CD44 extracellular domain by high-molecular-weight hyaluronan leads to recruitment of the polarity-regulating kinase PAR1b by the CD44 intracellular domain, which results in disruption of the Hippo signaling-inhibitory PAR1b-MST complex. Once liberated from PAR1b, MST activates Hippo signaling. Conversely, low-molecular-weight hyaluronan, which is produced by hyaluronidase-mediated degradation of high-molecular-weight hyaluronan, inhibits Hippo signaling by competing with high-molecular-weight hyaluronan for CD44 binding. Triple-negative breast cancers with higher hyaluronidase-2 expression show poorer prognosis than those with lower hyaluronidase-2 expression. Consistently, decreased hyaluronidase-2 is associated with reduced tumorigenicity in a tumor xenograft model. Hence, perturbation of high-molecular-weight hyaluronan-mediated Hippo signaling activation contributes to cancer aggressiveness.

More than 50% of people in the world are infected with Helicobacter pylori (H. pylori), which induces various gastric diseases. Especially, epidemiological studies have shown that H. pylori infection is a major risk factor for gastric cancer. It has been reported that the levels of interleukin (IL)-1β are upregulated in gastric tissues of patients with H. pylori infection. In this study, we investigated the induction mechanism of IL-1β during H. pylori infection. We found that IL-1βmRNA and protein were induced in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells after H. pylori infection. This IL-1β production was inhibited by a caspase-1 inhibitor and a ROS inhibitor. Furthermore, K(+) efflux and Ca(2+) signaling were also involved in this process. These data suggest that NOD-like receptor (NLR) family, pyrin domain containing 3 (NLRP3) and its complex, known as NLRP3 inflammasome, are involved in IL-1β production during H. pylori infection because it is reported that NLRP3 inflammasome is activated by ROS, K(+) efflux and/or Ca(2+) signaling. These findings may provide therapeutic strategy for the control of gastric cancer in H. pylori-infected patients.