**6. Cloning the MG** *COX-2* **promoter region**

As mentioned above, human *COX-2* has abundant CGIs in the promoter region. To examine whether CGIs are present in the *COX-2* promoter region of MGs, we performed genomic PCR with primers designed using as previously reported MG cDNA (Accession #: AB177842) [33]. The PCR product has an intron sequence in the promoter region. Then, gene walking was performed using a primer in this intron. The promoter region of the MG *COX-2* gene was obtained (**Figure 6**), and the sequence contains more CGIs than the mouse genome, and is comparable to that of the human genome. Pyrosequencing showed several methylated CGIs (>15%) in the MGC2 cells. Treatment with 5-aza-dC decreased the methylation levels in these cells (**Figure 7**). As was observed in the human stomach, *COX-2* methylation levels were increased in *H. pylori*-infected gastric mucosa of MGs when compared to the levels in

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**Figure 7.** Effect of 5-aza-dC treatment on *COX-2* methylation levels in MGC2 cells. Cells were treated with vehicle (A) with 5-aza-dC (B, 1 μmol/L) for 5 days, and then methylation levels of *COX-2* were analyzed by quantitative bisulfitepyrosequencing methods as previously reported [42]. Bisulfite treatment of gDNA was performed with the EpiTect bisulfite kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. Bisulfite-treated DNA (1 μL) was used as a template in subsequent PCR experiments. For most assays, we used touchdown PCR. All PCR assays included a denaturation step at 95°C for 30 s, followed by annealing at various temperatures for 30 s, and extension at 72°C for 30 s. After PCR, the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primers. Pyrosequencing was performed using PSQ HS 96 Gold single-nucleotide polymorphism reagents on a PyroMark Q24 pyrosequencing machine (QIAGEN). The protocol for pyrosequencing was described in detail previously [42]. Pyrosequencing quantitatively measures the methylation status of CpG sites in a target region. Adjacent sites usually show highly concordant methylation. Therefore, the mean percent methylation in detected sites can be used as a representative value for each gene promoter. Cases with the methylation density > 15% were regarded as methylation positive. Forward primer: tgggtgaggggaattttataga, reverse primer: aaaccctaaccatccttacaa;

the control mucosa (**Figure 8**).

and sequencing primer: aggagtttgtttaggaag.

**Figure 6.** Human, MG, and mouse *COX-2* promoter regions. Genome walking to isolate genomic, the *COX-2* promoter region of MGs, was performed by using the Straight Walk Kit [41] according to the manufacturer's instructions (Bex, Tokyo, Japan). Amplified fragments were cloned into the pSTBlue-1 vector (Novagen, Madison, WI, USA) and sequenced. More abundant CGIs were observed in the MG genome when compared to the mouse genome, and the number was comparable to human genome.

is comparable to that of the human genome. Pyrosequencing showed several methylated CGIs (>15%) in the MGC2 cells. Treatment with 5-aza-dC decreased the methylation levels in these cells (**Figure 7**). As was observed in the human stomach, *COX-2* methylation levels were increased in *H. pylori*-infected gastric mucosa of MGs when compared to the levels in the control mucosa (**Figure 8**).

PCR with primers designed using as previously reported MG cDNA (Accession #: AB177842) [33]. The PCR product has an intron sequence in the promoter region. Then, gene walking was performed using a primer in this intron. The promoter region of the MG *COX-2* gene was obtained (**Figure 6**), and the sequence contains more CGIs than the mouse genome, and

**Figure 6.** Human, MG, and mouse *COX-2* promoter regions. Genome walking to isolate genomic, the *COX-2* promoter region of MGs, was performed by using the Straight Walk Kit [41] according to the manufacturer's instructions (Bex, Tokyo, Japan). Amplified fragments were cloned into the pSTBlue-1 vector (Novagen, Madison, WI, USA) and sequenced. More abundant CGIs were observed in the MG genome when compared to the mouse genome, and the

number was comparable to human genome.

214 Chromatin and Epigenetics

**Figure 7.** Effect of 5-aza-dC treatment on *COX-2* methylation levels in MGC2 cells. Cells were treated with vehicle (A) with 5-aza-dC (B, 1 μmol/L) for 5 days, and then methylation levels of *COX-2* were analyzed by quantitative bisulfitepyrosequencing methods as previously reported [42]. Bisulfite treatment of gDNA was performed with the EpiTect bisulfite kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's protocol. Bisulfite-treated DNA (1 μL) was used as a template in subsequent PCR experiments. For most assays, we used touchdown PCR. All PCR assays included a denaturation step at 95°C for 30 s, followed by annealing at various temperatures for 30 s, and extension at 72°C for 30 s. After PCR, the biotinylated strand was captured on streptavidin-coated beads (Amersham Bioscience, Uppsala, Sweden) and incubated with sequencing primers. Pyrosequencing was performed using PSQ HS 96 Gold single-nucleotide polymorphism reagents on a PyroMark Q24 pyrosequencing machine (QIAGEN). The protocol for pyrosequencing was described in detail previously [42]. Pyrosequencing quantitatively measures the methylation status of CpG sites in a target region. Adjacent sites usually show highly concordant methylation. Therefore, the mean percent methylation in detected sites can be used as a representative value for each gene promoter. Cases with the methylation density > 15% were regarded as methylation positive. Forward primer: tgggtgaggggaattttataga, reverse primer: aaaccctaaccatccttacaa; and sequencing primer: aggagtttgtttaggaag.

*H. pylori*-infected gastric mucosa. In addition, *COX-2* promoter methylation levels in patients with successfully eradicated *H. pylori* infection are lower than the levels in *H. pylori*-positive cases. As mentioned above, experiments using rodents have shown that *COX-2* mRNA expression is enhanced during ulcer healing. Thus, we investigated the influence of *H. pylori* infection on COX-2 expression during stomach ulcer healing in MGs. Acetic acid-induced ulcer healing was delayed in *H. pylori*-infected MG stomachs when compared to the healing of ulcers without *H. pylori* infection [29]. The *COX-2* promoter region of MGs also has abundant CGIs, comparable to human *COX-2*. Thus, MGs are good models for investigating the role of *COX-2* methylation in gastric mucosal healing. While *COX-2* mRNA expression at the ulcer edge was increased 5 days after acetic acid injection in MG stomachs without *H. pylori* infection, as was observed in the mouse and rat models, such increases in *COX-2* mRNA expression were not observed in *H. pylori*-infected MG gastric mucosa. However, a demethylating agent restored COX-2 mRNA expression in both human and MG gastric carcinoma cell lines in which COX-2 is densely methylated. Accordingly, we demonstrated *in vitro* and *in vivo* that *COX-2* mRNA expression is regulated through an epigenetic mechanism in human and MG gastric mucosa. Aberrant DNA methylation has been extensively investigated in the context of the pathogenesis of various cancers, including stomach cancer. In addition, the involvement of epigenetic modifications has been reported in the pathogenesis of various chronic diseases, such as essential hypertension and cardiovascular disease [39]. Taken together, the epigenetic inhibition of COX-2 expression due to chronic inflammation induced by *H. pylori* infection seems to interfere with ulcer healing and increase the vulnerability of the gastric mucosa. The decrease in *COX-2* methylation levels after *H. pylori* eradication may restore

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*H. pylori* infection, the leading cause of peptic ulcer disease, induces sustained inflammation in the gastric mucosa. This chronic inflammation causes aberrant DNA methylation in various genes. The *COX-2* promoter regions in both MGs and humans contain abundant CGIs. COX plays critical roles in peptic ulcer development and healing, and both COX-1 and COX-2 are necessary for the gastric mucosal defense system. COX-1 is a housekeeping enzyme, which maintains microcirculation and mucous production. During ulcer healing, COX-2 is expressed at the margin of an ulcer, and COX-2-derived PGs induce various growth factors to promote mucosal healing and angiogenesis. Ulcer healing is delayed in *H. pylori* infected-MG stomachs, which is the best model of *H. pylori* infection in humans. We showed that *COX-2* mRNA induction during ulcer healing was impaired in *H. pylori*-infected-MG stomachs. The *COX-2* promoter region is methylated in both *H. pylori*-infected human and MG gastric mucosa. These CGIs are also methylated in human Kato III cells and MGC2 gastric carcinoma cells. *COX-2* mRNA expression in these cells is restored by treatment with a demethylating agent. In *H. pylori*-infected gastric mucosa, *COX-2* promoter methylation appears to be involved in the impaired *COX-2* mRNA induction typically observed during ulcer healing,

gastric mucosal defense.

which leads to delayed ulcer healing.

**8. Conclusions**

**Figure 8.** DNA methylation levels at *COX-2* in the MG gastric mucosa in the presence or absences of *H. pylori* infection. Methylation levels at *COX-2* in the MG gastric mucosa were analyzed by quantitative bisulfite pyrosequencing.


**3.3. SNP-based sequencing**

This third approach was the most recent method introduced to the variety of clinically available NIPT options. This technique involves targeted amplification and sequencing of singlenucleotide polymorphisms (SNPs). SNPs are single base pairs that occur approximately once / 300 base pairs on the human genome and can be used to distinguish individuals. In addition to the above mentioned applications, maternal and fetal DNA also can be distinguished by SNP analysis. For this analysis, both maternal DNA from white cells from the buffy coat and maternal plasma which includes fetal and maternal DNA are used. In the SNP-technology originally introduced by Zimmermann et al. [16], 19,488 SNPs on the chromosomes 21, 13, 18, X, and Y are analyzed simultaneously. Taking into account the parental genotype, the fetal fraction, and the fetal chromosome copy number, billions of possible genotypes at a specific locus are considered by a complex algorithm and the observed allele distributions are com‐ pared to the expected allele distributions. By this method, the most likely fetal genotype can be calculated and a specific risk score for the analyzed aneuploidies is reported [16–19].


**6. Acknowledgement**

The supports from Thailand Research Fund and National Metal and Materials Technology Center (MTEC), an organization under National Science and Technology Development Agency (NSTDA), are gratefully acknowledged. The authors also would like to thank Dr. John T.H. Pearce for language assistant.


**Acknowledgements**

The author expresses a deep gratitude to the young colleagues Ievgen Zaitsev, Borys Jeleński, and Jan Piekarski, as well as to the colleague Henryk Chaciñski for their active participation in the elaboration of the AFCS prototype and creative engineering thinking.


**Featured Application: Structure health monitoring for refinery equipment, bridges, rotating machines, cranes and draglines.**

**Abstract:** Acoustic emission (AE) testing is traditionally carried out on non-operating objects. Such requirement is associated both with the Kaiser effect, leading to the necessity for exceeding the test load above the working one and with a high level of noise during object operation. However, AE testing could be performed under operating conditions, if the AE data acquisition period is increased and a specialized method is developed, which should take account of the effect of various noise, features of the object loading under operating conditions, the effect of damaging factors and possible destruction mechanisms. This paper investigates the possibility to carry out structural health monitoring (SHM) of hydrotreaters, highway bridges, high-temperature pipelines, gas adsorbers, roller bearings of rotary kilns and draglines on the basis of AE method. Architecture of SHM-system and specific data analysis procedures are proposed.

**Keywords:** structural health monitoring; acoustic emission; non-destructive testing; hydrotreater; bridge; high temperature; gas adsorber; rotary kiln; dragline


1. Introduction

In devices for thermonuclear fusion research, for example, of the Tokamak type, particles of hydrogen isotopes, deuterium and tritium, are in the form of a hot fully ionized plasma [1, 2]. To avoid a destruction of the machine wall, a special region, the so-called scrape-off layer (SOL), is arranged at the plasma edge, where particles stream along the magnetic field to the

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

special target plates [3]. Normally, it is done by using additional magnetic coils to form a divertor configuration (see Figure 1a).

permitting the convergence of iterations to the error level defined by the machine accuracy, this method, nonetheless, is also time-consuming. The reason is the necessity to assess integrals in the velocity space from functions involving the ion velocity distribution function, additionally to integrations in the normal space. Recently [4], an approximate pass method has been applied to evaluate these integrals, and the acceleration of kinetic calculations by a factor of

Noise-Free Rapid Approach to Solve Kinetic Equations for Hot Atoms in Fusion Plasmas

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The amendments, outlined above, have allowed to perform calculations of the plasma parameters in the DEMO SOL with cx atoms described kinetically, by varying the input parameters, for example, the plasma transport characteristics, in a broad range [4]. In addition the results of these computations have been thoroughly compared with those obtained with cx species described in the so-called diffusion approximation. This approximation, often used in diverse edge modeling approaches to save CPU time (see, e.g., [7, 8]), is strictly valid under plasma conditions of low temperature and high density where the time between cx collisions of atoms with ions is much smaller than that till their ionization by electrons. Across the DEMO SOL, the plasma density and

The usage of a diffusion approximation for cx atoms, generated from species recycling from the wall, becomes especially questionable by considering the situation near an opening in the vessel wall. Such openings will be made in a reactor for diverse purposes, for example, for ducts leading to the first mirrors, collecting light emitted by impurity species in the plasma (see Figure 1b). These installations are inaccessible for charged plasma particles, moving mostly along magnetic field lines and penetrating into the duct at a distance of 1 mm. However, hot cx atoms, unconfined by the magnetic field, can freely hit and erode these installations. The erosion rate is very sensitive to the energy spectrum of cx atoms which can be significantly dependent on the modeling approach. At the position of the duct opening, the inflow of recycling neutrals is actually absent, and the transfer of cx atoms has two- or even threedimensional pattern. By calculating the density of cx atoms in the vicinity of a circular opening from a 2D diffusion equation, the erosion rate of a first mirror of Mo has been assessed in [9]. In the present paper, we extend the approaches, elaborated in [4] to model cx atoms kinetically in

The results are compared with those of [9]. Although there cx atoms have been considered in the diffusion approximation to reduce CPU time, the new approach allows to perform more

Although the concept of magnetic fusion is based on the idea that charged particles can be infinitely long confined within closed magnetic surfaces, there are diverse physical mechanisms leading to the losses across these surfaces (see, e.g., [10]). Therefore, electrons and ions escape through the separatrix into the SOL and may reach the first wall of the machine vessel.

the temperatures of electrons and ions change, however, by orders of magnitude [4].

50 has been achieved.

the 1D case, on a 2D geometry.

2. Basic equations

exact kinetic calculations even by orders of magnitude faster.

By reaching the divertor target plates, plasma electrons and ions are recombined into neutral atoms and molecules which are finally exhausted from the device by pumps. However, in a future fusion reactor like DEMO [1, 2], only a minor fraction of 1% of neutrals generated at the targets will be pumped out. The rest of them is ionized again in the plasma near the targets. This "recycling" process significantly restrains the parallel plasma flow in the SOL [4]. Therefore, a considerable fraction of plasma particles lost from the plasma core will reach the vessel wall before they are exhausted into the divertor. Plasma fluxes to the wall saturate it with fuel particles in a time much shorter than the discharge duration, and a comparable amount of neutral species will recycle back from the wall into the plasma. Recycling neutrals are not confined by the magnetic field and penetrate at several centimeters into the SOL. Here, charge exchange (cx) collisions of them with ions generate atoms of energies much higher than that of primary recycling neutral particles. A noticeable fraction of such secondary cx atoms hit the vessel wall and erode it.

Statistical Monte Carlo methods [5] are normally used to model cx atoms at the edge of fusion devices. A crucial obstacle to apply these approaches for extensive parameter studies, for example, with the aim to optimize the duct geometry, has too long calculations needed to achieve reasonably small accident errors. This is, however, necessary, for example, to couple neutral parameters with the plasma calculations. In a one-dimensional geometry, an alternative approach, based on iteration procedure to solve the kinetic equation represented in an integral form, has been elaborated decades ago [6]. Being free from statistical noise and

Figure 1. The cross section of toroidally symmetric fusion device of the Tokamak type with the SOL region formed by the presence of a divertor (a) and the processes near the opening in the wall for a duct guiding to a first mirror (b).

permitting the convergence of iterations to the error level defined by the machine accuracy, this method, nonetheless, is also time-consuming. The reason is the necessity to assess integrals in the velocity space from functions involving the ion velocity distribution function, additionally to integrations in the normal space. Recently [4], an approximate pass method has been applied to evaluate these integrals, and the acceleration of kinetic calculations by a factor of 50 has been achieved.

The amendments, outlined above, have allowed to perform calculations of the plasma parameters in the DEMO SOL with cx atoms described kinetically, by varying the input parameters, for example, the plasma transport characteristics, in a broad range [4]. In addition the results of these computations have been thoroughly compared with those obtained with cx species described in the so-called diffusion approximation. This approximation, often used in diverse edge modeling approaches to save CPU time (see, e.g., [7, 8]), is strictly valid under plasma conditions of low temperature and high density where the time between cx collisions of atoms with ions is much smaller than that till their ionization by electrons. Across the DEMO SOL, the plasma density and the temperatures of electrons and ions change, however, by orders of magnitude [4].

The usage of a diffusion approximation for cx atoms, generated from species recycling from the wall, becomes especially questionable by considering the situation near an opening in the vessel wall. Such openings will be made in a reactor for diverse purposes, for example, for ducts leading to the first mirrors, collecting light emitted by impurity species in the plasma (see Figure 1b). These installations are inaccessible for charged plasma particles, moving mostly along magnetic field lines and penetrating into the duct at a distance of 1 mm. However, hot cx atoms, unconfined by the magnetic field, can freely hit and erode these installations. The erosion rate is very sensitive to the energy spectrum of cx atoms which can be significantly dependent on the modeling approach. At the position of the duct opening, the inflow of recycling neutrals is actually absent, and the transfer of cx atoms has two- or even threedimensional pattern. By calculating the density of cx atoms in the vicinity of a circular opening from a 2D diffusion equation, the erosion rate of a first mirror of Mo has been assessed in [9]. In the present paper, we extend the approaches, elaborated in [4] to model cx atoms kinetically in the 1D case, on a 2D geometry.

The results are compared with those of [9]. Although there cx atoms have been considered in the diffusion approximation to reduce CPU time, the new approach allows to perform more exact kinetic calculations even by orders of magnitude faster.