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Introduction: Risk prediction of gastric cancers is important to implement appropriate screening procedures. Although aberrant DNA methylation is deeply involved in gastric carcinogenesis, its induction by Helicobacter pylori, a strong gastric carcinogen, is unclear. Here, we analyzed the effect of H. pylori infection on the quantity of methylated DNA molecules in noncancerous gastric mucosae and examined its association with gastric cancer risk.Experimental Design: Gastric mucosae were collected from 154 healthy volunteers (56 H. pylori negative and 98 H. pylori positive) and 72 cases with differentiated-type gastric cancers (29 H. pylori negative and 43 H. pylori positive) by endoscopy. The numbers of DNA molecules methylated and unmethylated for eight regions of seven CpG islands (CGI) were quantified by quantitative PCR after bisulfite modification, and fractions of methylated molecules (methylation levels) were calculated.Results: Among healthy volunteers, methylation levels of all the eight regions were 5.4- to 303-fold higher in H. pylori positives than in H. pylori negatives (P < 0.0001). Methylation levels of the LOX, HAND1, and THBD promoter CGIs and p41ARC exonic CGI were as high as 7.4% or more in H. pylori–positive individuals. Among H. pylori–negative individuals, methylation levels of all the eight regions were 2.2- to 32-fold higher in gastric cancer cases than in age-matched healthy volunteers (P ≤ 0.01). Among H. pylori–positive individuals, methylation levels were highly variable, and that of only HAND1 was significantly increased in gastric cancer cases (1.4-fold, P = 0.02).Conclusions: It was indicated that H. pylori infection potently induces methylation of CGIs to various degrees. Methylation levels of specific CGIs seemed to reflect gastric cancer risk in H. pylori–negative individuals.

Supplementary Table S1 from High Levels of Aberrant DNA Methylation in Helicobacter pylori–Infected Gastric Mucosae and its Possible Association with Gastric Cancer Risk

The interfacial microstructure and hardness of cladding plates produced by explosive welding between magnesium alloys having different aluminum concentrations and A6005C aluminum alloy were investigated. Further, measurements of residual stress at the interface of cladding plates were performed. In all cladding plates, the bonding interface had a wavy shape. Adiabatic shear bands were formed at the interface on the magnesium alloy side and deformation twins appeared at the interface due to the impact of explosive welding. Microstructure observation using scanning transmission electron microscope revealed that a thin interlayer was formed at the interface in all cladding plates. The thickness of the interlayer increased with an increase in aluminum concentration in the magnesium alloy, while the thickness was 1 μm or less. In the cross-section of the cladding plate, aluminum alloy showed a relatively higher Vickers hardness value compared with the magnesium alloy, and the hardness value increased when approaching the interface. However, nanoindentation tests revealed no increase in hardness was observed at the interface. Measurements of the residual stress using synchrotron radiation x-rays at the interface of cladding plates revealed a tendency for the occurrence of tensile residual stress on the magnesium alloy side and compressive residual stress on the aluminum alloy side. This might be due to a difference in the coefficient of thermal expansion between the magnesium and aluminum alloys.

The scope of this work was to investigate the quench sensitivity of a high-purity wrought aluminum alloy Al6Zn0.75 Mg (in this work called 7003pure). This is compared to a similar alloy with the additions of Fe, Si, and Zr at a sum less than 0.3 at.% (in this work called 7003Fe,Si,Zr). Differential scanning calorimetry (DSC) was used for an in situ analysis of quench induced precipitation in a wide range of cooling rates varying between 0.0003 and 3 K/s. In 7003pure, three main precipitation reactions were observed during cooling, a medium temperature reaction with a distinct double peak between 325 and 175 °C and a very low temperature reaction starting at about 100 °C. An additional high temperature reaction related to the precipitation of Mg2Si starting at 425 °C has been observed for 7003Fe,Si,Zr. In terms of hardness after natural as well as artificial aging, alloy 7003pure shows a very low quench sensitivity. Hardness values on the saturation level of about 120 HV1 are seen down to cooling rates of 0.003 K/s. The as-quenched hardness (5 min of natural aging) shows a maximum at a cooling rate of 0.003 K/s, while slower and faster cooling results in a lower hardness. In terms of hardness after aging, 0.003 K/s could be defined as the technological critical cooling rate, which is much higher for 7003Fe,Si,Zr (0.3–1 K/s). The physical critical cooling rates for the suppression of any precipitation during cooling were found to be about 10 K/s for both variants.

Explosive welding can be used to join materials using controlled explosions, creating materials with desirable properties. Assistant Professor Mami Mihara-Narita, Department of Physical Science and Engineering, Nagoya Institute of Technology (NIT), Japan, is exploring the potential of joining technology by explosive welding for the multi-materialism of lightweight materials such as magnesium alloy and aluminium alloy and evaluating their characteristics for practical use. She hopes that her work will contribute to the weight reduction of transportation materials, as well as benefiting transportation equipment and clean energy materials. Mihara-Narita is collaborating closely with Professor Yoshimi Watanabe and Associate Professor Hisashi Sato from NIT, and Dr Yasumasa Chino and Dr Hisashi Mori from the National Institute of Advanced Industrial Science and Technology (AIST and UACJ corporation) in this work. The team is also performing characteristic analysis of explosive welded materials, using a transmission electron microscope to observe interfacial microstructure. The researchers are also evaluating joint strength by shear test, measuring hardness and micro deformation by nanoindentation method and measuring residual stress by X-ray diffraction and neutron diffraction method. In addition they are investigating fatigue strength in order to ensure materials are suitable for use in transportation equipment. Fatigue strength will continue to be a key focus for the researchers moving forward, and they will also be performing data analysis of their data on explosive welding conditions, metallographic structure and various properties of explosive welding clad material.

Two high-purity aluminium alloys based on composition Al–3.0Mg–1.0Cu (wt%), one with added 0.4 wt% Ag, were compared up to 11 days ageing at 443 K by means of transmission electron microscopy and hardness measurements. The base alloy exhibits an inhomogeneous precipitate microstructure with a high density of fine needle-shaped Guinier-Preston-Bagaryatsky (GPB) zones together with coarser precipitates of S′-Al2CuMg and rods of the structurally unknown Z-phase. The S′ phase is preferably formed on dislocations. The addition of Ag has a strong effect, leading to a homogeneous distribution with fine Ag-containing icosahedral quasi-crystalline precipitates (iQC). Both the GPB zones in the base alloy and the iQC phase in the Ag added alloy survive even after long term ageing. Ag is found to suppress the formation of the S′ phase. It is suggested that the Z phase is an approximation phase to the quasi-crystalline phase as is the case for the T-phase, implying they are based on similar (Bergman) clusters. This is a submitted manuscript of an article published by Elsevier Ltd in Materials Science and Engineering: A, 27 January 2016.

The effects of the pre-aging period on the formation behavior of nanoclusters during two-step aging in an Al-3.0Mg-1.0Cu (mass%) alloy has been investigated by the hardness, differential scanning calorimetry (DSC) and electrical resistivity measurement. Two-step aging which consists of pre-aging and final aging results in an increase of the peak hardness. When the pre-aging is conducted at 323K for 1.2ks, the hardness dramatically increases after the final aging treatment. This pre-aging period corresponds to the incubation stage which is one of the hardening stages in an Al-Mg-Cu alloy. During the pre-aging, no clear hardness and electrical resistivity increase are obtained, however a certain structural change is considered to occur. In this study, co-clusters are assumed to be formed during pre-aging. On the contrary, when the preaging period is 345.6ks, the hardness and electrical resistivity decrease in the beginning of final aging. It is attributed to the reversion of nanoclusters. In this case, the pre-aging period corresponds to the first stage of hardening. The first stage of hardening, which occurs rapidly (e.g. within 60s at 443K) and contributes 60% of the total hardening, is attributed to solute clustering rather than to the formation of GPB zones. [doi:10.2320/matertrans.L-M2015801]

The characteristic age-hardening response of Al-3.0Mg-1.0Cu (mass%) alloys with and without Ag addition has been investigated by the hardness measurement, differential scanning calorimetry (DSC) and electrical resistivity measurement. The alloy compositions locating in the (α+S+T) phase field of the Al-Mg-Cu phase diagram are known to be effective to harden in two stages separated by a distinct and often prolonged hardness plateau. The first stage of hardening occurs very rapidly (e.g. within 60 s at 443 K) and contributes to increase hardness as much as 50 % of the total age-hardening. In the Ag-added alloy, the hardness change during the first stage of hardening is larger and the plateau stage is shortened as a result of the fast arrival at the second stage of hardness. Small amount of Ag changes the age hardening response of the Base alloy dramatically. In the low temperature aging, the incubation stage at which no clear hardness and electrical resistivity increase appears for a long period before the first stage of hardening. After the pre-aging at this incubation period, a characteristic two-step aging response is observed. The peak hardness dramatically changes in the Al-3.0Mg-1.0Cu alloy, while no clear change in the Ag-added alloy.

Effect of Ag addition on the age-hardening response and precipitate microstructure of the Al-3.0Mg-1.0Cu (wt%) alloy has been investigated by micro Vickers hardness and transmission electron microscopy (TEM). The alloy compositions located in the (α+S+T) phase field of the Al-Mg-Cu phase diagram are known to be effective to harden in two stages separated by a distinct and often prolonged hardness plateau. The first stage of hardening occurs very rapidly (e.g. within 60 s at 443 K) and contributes to increase the hardness as much as 50% of the total age-hardening. Trace addition of Ag changes the age hardening response of the Al-3.0Mg-1.0Cu alloy dramatically. In the Ag-added alloy, the hardness change during the first stage of hardening is larger and the plateau stage is shortened as a result of the fast arrival at the second stage of hardness. The microstructure at the plateau stage of hardening for the Al-3.0Mg-1.0Cu alloy exhibits no clear evidence of any precipitates contributing to the first rapid hardening. On the other hand, lath-shaped S phase was observed at the peak hardness. In the Ag-added alloy, fine distributed precipitates were obtained from the beginning of age-hardening.

Gene silencing by methylation of promoter CpG islands is deeply involved in cancers, but its involvement in polyclonal disorders is still unclear. Here, we analyzed the presence of gene silencing in intestinal metaplasia (IM) of the stomach, a polyclonal disorder, in which multiple gastric glands aberrantly differentiate into those with intestinal characteristics. By a genome-wide screening, CpG islands in the putative promoter regions of four genes (ZIK1, ZNF141, KAL1, and FGF14) were found to be specifically methylated in glands with IM, and their expression was markedly decreased. When demethylation was induced in cell lines with their methylation by 5-aza-2′-deoxycytidine, expression of ZIK1, KAL1, and FGF14 was restored, supporting causal roles of methylation in their silencing. Analysis of ZIK1 methylation in a single gland showed that the vast majority of DNA molecules isolated from a gland with IM were methylated and that those from a gland without IM were not. ZIK1 methylation was present in glands isolated from physically distant positions within a stomach, showing that methylation occurred multifocally. These data indicate that methylation of multiple genes occurs independently in multiple glands, each of which has its own stem cell, demonstrating that involvement of aberrant gene silencing in noninherited polyclonal human disorders needs more attention.

Aims: Other than ectopic expression of intestinal transcription factors, Cdx1 and Cdx2, the molecular mechanisms underlying gastric and intestinal phenotypes of human stomach adenocarcinomas have yet to be clarified in detail. We have reported that Sox2, an HMG-box gastric transcription factor, is expressed in normal gastric mucosa and down-regulated in intestinal metaplasia. Methods and results: We analysed mRNA levels of Sox2 and other differentiation markers in 50 surgically resected stomach adenocarcinomas, immunohistochemically classified into gastric (G), gastric-and-intestinal (GI)-mixed, solely intestinal (I), and null (N) types. Sox2 was found to be transcribed in G and GI-mixed type adenocarcinomas in accordance with MUC5AC and MUC6 expression, while Cdx1 and Cdx2 were up-regulated in GI-mixed and I types along with the expression of MUC2 and villin. In the N type, both gastric and intestinal transcription factors were suppressed. Immunohistochemistry confirmed expression of Sox2 in MUC5AC+ lesions and Cdx2 localization together with MUC2. A stomach adenocarcinoma cell line, KATOIII, demonstrated both MUC5AC and Sox2, although MUC5AC mRNA was not detected in the Sox2+ AGS cell line. Conclusions: Sox2 may play an important role in maintaining a gastric phenotype in stomach cancers as well as in normal tissue, in cooperation with other cofactor(s).

Gene silencing by methylation of promoter CpG islands is deeply involved in cancers, but its involvement in polyclonal disorders is still unclear. Here, we analyzed the presence of gene silencing in intestinal metaplasia (IM) of the stomach, a polyclonal disorder, in which multiple gastric glands aberrantly differentiate into those with intestinal characteristics. By a genome-wide screening, CpG islands in the putative promoter regions of four genes (ZIK1, ZNF141, KAL1, and FGF14) were found to be specifically methylated in glands with IM, and their expression was markedly decreased. When demethylation was induced in cell lines with their methylation by 5-aza-2′-deoxycytidine, expression of ZIK1, KAL1, and FGF14 was restored, supporting causal roles of methylation in their silencing. Analysis of ZIK1 methylation in a single gland showed that the vast majority of DNA molecules isolated from a gland with IM were methylated and that those from a gland without IM were not. ZIK1 methylation was present in glands isolated from physically distant positions within a stomach, showing that methylation occurred multifocally. These data indicate that methylation of multiple genes occurs independently in multiple glands, each of which has its own stem cell, demonstrating that involvement of aberrant gene silencing in noninherited polyclonal human disorders needs more attention.

Introduction: Risk prediction of gastric cancers is important to implement appropriate screening procedures. Although aberrant DNA methylation is deeply involved in gastric carcinogenesis, its induction by Helicobacter pylori, a strong gastric carcinogen, is unclear. Here, we analyzed the effect of H. pylori infection on the quantity of methylated DNA molecules in noncancerous gastric mucosae and examined its association with gastric cancer risk. Experimental Design: Gastric mucosae were collected from 154 healthy volunteers (56 H. pylori negative and 98 H. pylori positive) and 72 cases with differentiated-type gastric cancers (29 H. pylori negative and 43 H. pylori positive) by endoscopy. The numbers of DNA molecules methylated and unmethylated for eight regions of seven CpG islands (CGI) were quantified by quantitative PCR after bisulfite modification, and fractions of methylated molecules (methylation levels) were calculated. Results: Among healthy volunteers, methylation levels of all the eight regions were 5.4- to 303-fold higher in H. pylori positives than in H. pylori negatives (P < 0.0001). Methylation levels of the LOX, HAND1, and THBD promoter CGIs and p41ARC exonic CGI were as high as 7.4% or more in H. pylori–positive individuals. Among H. pylori–negative individuals, methylation levels of all the eight regions were 2.2- to 32-fold higher in gastric cancer cases than in age-matched healthy volunteers (P ≤ 0.01). Among H. pylori–positive individuals, methylation levels were highly variable, and that of only HAND1 was significantly increased in gastric cancer cases (1.4-fold, P = 0.02). Conclusions: It was indicated that H. pylori infection potently induces methylation of CGIs to various degrees. Methylation levels of specific CGIs seemed to reflect gastric cancer risk in H. pylori–negative individuals.

The molecular mechanisms underlying the development of intestinal metaplasia (IM) of the human stomach have yet to be clarified in detail. Besides ectopic expression of intestinal transcription factors, Cdx1 and Cdx2, little information is available regarding other regulatory factors. Hence, we here analyzed Sox2, a human homolog of a chicken gastric transcription factor, with reference to our new classification for gastric/intestinal (GI)-mixed type IM. Twenty specimens of surgically resected antral mucosa were subjected to a gland isolation technique. Isolated glands were classified into gastric (G), GI-mixed, and solely intestinal (I) types according to Alcian blue and paradoxical concanavalin A staining and were quantified for mRNA levels of gastrointestinal markers. MUC5AC and MUC6 transcripts decreased with the progression of IM, while MUC2 and villin-1 were inversely correlated. Sox2 showed a gradual decrease from G, through GI, to the I type (G vs GI and GI vs I, P

To evaluate the genetic factors of familial predisposition to gastric cancer, genetic alterations in the surgically resected stomach samples from gastric-cancer-prone families were investigated. Familial gastric cancer (FGC) was defined as gastric cancer occurring in a family with 3 or more gastric cancer patients over at least two successive generations. We examined replication error (RER) of six microsatellite markers and screened mutations of the 10-(A) repeat sequence in the transforming growth factor-beta receptor type II (TGF-betaRII) gene in individuals from seven unrelated FGC families. Three cases showed RER at one of the six (CA)n microsatellite markers but the other 4 cases showed no RER at any of these loci. No mutation was found in the 10-(A) repeat of the TGF-betaRII gene. Additionally, no germline mutation was found by polymerase chain reaction-single strand conformation polymorphism in exons 1-16 of E-cadherin, exons 5-8 of p53 and in the mutation cluster region of APC. These results indicate that disorders in the DNA mismatch repair system, E-cadherin, p53 and APC may be infrequently involved in the carcinogenesis of Japanese FGC.

The clinical features of familial gastric cancer are still unknown. To approach this question, we investigated the clinicopathological characteristics of 16 cases of familial gastric cancer. In this study the criteria used to define familial gastric cancer was the existence of three or more family members with gastric cancer in at least two successive generations. The clinicopathological characteristics of cases who fulfilled this criteria were studied. This study contained 16 familial gastric cancer probands. Seven cases (44%) of gastric cancer had developed at the cardiac region of the stomach. This frequency was significantly higher than for gastric cancer in the general population in Japan (15.4%, p < 0.01). Undifferentiated types were dominant in familial gastric cancer (69%, p < 0.05). Furthermore, the frequency of disseminated peritoneal (40%) and liver metastases (20%) in familial gastric cancer was also significantly higher than for gastric cancer in the general population in Japan (10.9%, p < 0.01, and 4.4%, p < 0.05, respectively). Familial gastric cancers were frequently located at the cardiac region and appeared to be more aggressive than sporadic gastric cancers. The unique characteristics of familial gastric cancer suggest a genetic background in their etiology.