An integrative approach for exploring the nature of fibroepithelial neoplasms.

BACKGROUND: Malignant phyllodes tumour (MPT) is a rare breast malignancy with epithelial and mesenchymal features. Currently, there are no appropriate research models or effective targeted therapeutic approaches for MPT. METHODS: We collected fresh frozen tissues from nine patients with MPT and performed whole-exome and RNA sequencing. Additionally, we established patient-derived xenograft (PDX) models from patients with MPT and tested the efficacy of targeting dysregulated pathways in MPT using the PDX model from one MPT. RESULTS: MPT has unique molecular characteristics when compared to breast cancers of epithelial origin and can be classified into two groups. The PDX model derived from one patient with MPT showed that the mouse epithelial component increased during tumour growth. Moreover, targeted inhibition of platelet-derived growth factor receptor (PDGFR) and phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) by imatinib mesylate and PKI-587 showed in vivo tumour suppression effects. CONCLUSIONS: This study revealed the molecular profiles of MPT that can lead to molecular classification and potential targeted therapy, and suggested that the MPT PDX model can be a useful tool for studying the pathogenesis of fibroepithelial neoplasms and for preclinical drug screening to find new therapeutic strategies for MPT.

Genomic characteristics of invasive mucinous adenocarcinoma of the lung with multiple pulmonary sites of involvement.

Invasive mucinous adenocarcinoma (IMA) of the lung frequently presents with diffuse pneumonic-type features or multifocal lesions, which are regarded as a pattern of intrapulmonary metastases. However, the genomics of multifocal IMAs have not been well studied. We performed whole exome sequencing on samples taken from 2 to 5 regions in seven patients with synchronous multifocal IMAs of the lung (24 regions total). Early initiating driver events, such as KRAS, NKX2-1, TP53, or ARID1A mutations, are clonal mutations and were present in all multifocal IMAs in each patient. The tumor mutational burden of multifocal IMAs was low (mean: 1.13/mega base), but further analyses suggested intra-tumor heterogeneity. The mutational signature analysis found that IMAs were predominantly associated with endogenous mutational process (signature 1), APOBEC activity (signatures 2 and 13), and defective DNA mismatch repair (signature 6), but not related to smoking signature. IMAs synchronously located in the bilateral lower lobes of two patients with background usual interstitial pneumonia had different mutation types, suggesting that they were double primaries. In conclusion, genomic evidence found in this study indicated the clonal intrapulmonary spread of diffuse pneumonic-type or multifocal IMAs, although they can occur in multicentric origins in the background of usual interstitial pneumonia. IMAs exhibited a heterogeneous genomic landscape despite the low somatic mutation burden. Further studies are warranted to determine the clinical significance of the genomic characteristics of IMAs in expanded cohorts.

Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells.

Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.

Investigating Self-Centering Capacity of Superelastic Shape Memory Alloy Fibers with Different Anchorages Through Pullout Tests.

This study investigated the pull-out resistance of superelastic shape memory alloy (SMA) short fibers in mortar with consideration of various end-anchorages that provide different anchoring actions. For the purpose, four types of SMA fibers were prepared using NiTi SMA wires with a diameter of 1.0 mm and the following four end shapes: straight (ST), L-shaped (LS), N-shaped (NS), and spearhead-shaped (SH). The straight-ended fiber was a reference with no working on the end, and the fiber with the spearhead-shaped end was crimped to make the end part flat. The fibers with L- and N-shaped ends were bent with single or double bending. The results showed that only the spearhead-shaped fibers showed self-centering behavior because of the superelasticity of the SMA after slip occurred. This paper discusses the reasons that the ST, LS, and NS fibers do not show self-centering behavior and proposes a concept to induce superelastic behavior in SMA fibers in mortar or concrete.

Dysregulated Wnt signalling and recurrent mutations of the tumour suppressor RNF43 in early gastric carcinogenesis.

Several recurrent mutations and epigenetic changes have been identified in advanced gastric cancer, but the genetic alterations associated with early gastric carcinogenesis and malignant transformation remain unclear. We investigated the genomic and transcriptomic landscape of adenomas with low-grade dysplasia (LGD) and high-grade dysplasia (HGD), and intestinal-type early gastric cancer (EGC). The results were validated in an independent cohort that included EGCs directly adjacent to adenoma (EGC-adenomas) that were in the process of malignant transformation, and de novo EGCs that do not seem to have been derived from adenoma. The expression patterns clearly divided into normal, LGD, and EGC, whereas those of HGD overlapped with LGD or EGC. These results suggest that HGD is the critical stage determining malignant transformation. We found that genes related to focal adhesion and extracellular matrix receptor interaction pathways were upregulated as LGD progressed to EGC, whereas canonical Wnt signalling and peroxisome proliferator-activated receptor (PPAR) signalling pathway genes were downregulated in EGC. Genomic alterations such as somatic mutation, gene fusion and copy number variation increased gradually from LGD to EGC. APC mutations were present in 67% of LGDs, 58% of HGDs, and 18% of EGCs. RNF43 mutations were present only in HGD and EGC, and TP53 mutations were present only in EGC. In a validation cohort, RNF43 mutations were present in 35.2% of EGC-adenomas, but in only 8.6% of de novo EGCs. This is the first study to investigate the genomic and transcriptomic landscape of multistep gastric carcinogenesis. We investigated important alterations and their related pathways in each step as tumours progressed from LGD to HGD and eventually to EGC. We suggest that mutations and downregulation of RNF43 may play a critical role in the transition from adenoma to carcinoma. Given these findings and Wnt dependency in tumours with RNF43 mutation, intestinal-type gastric cancer or adenoma with RNF43 mutation might represent a promising indication for Wnt-targeted agents. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Deregulation of immune response genes in patients with Epstein-Barr virus-associated gastric cancer and outcomes.

BACKGROUND & AIMS: Patients with Epstein-Barr virus-associated gastric carcinoma (EBVaGC) have a better prognosis than those with gastric cancer not associated with EBV infection (EBVnGC). This is partly because EBV infection recruits lymphocytes, which infiltrate the tumor. A high degree of tumor heterogeneity is likely to be associated with poor response. We investigated differences in gene expression patterns between EBVaGC and EBVnGC. METHODS: We used gene expression profile analysis to compare tumor and nontumor gastric tissues from 12 patients with EBVaGC and 14 patients with EBVnGC. Findings were validated by whole transcriptome RNAseq and real-time quantitative polymerase chain reaction analyses. CD3(+) primary T cells were isolated from human blood samples; migration of these cells and of Jurkat cells were measured in culture with EBV-infected and uninfected gastric cancer cells. RESULTS: Based on Pearson correlation matrix analysis, EBVaGCs had a higher degree of homogeneity than EBVnGCs. Although 4550 genes were differentially expressed between tumor and nontumor gastric tissues of patients with EBVnGC, only 186 genes were differentially expressed between tumor and nontumor gastric tissues of patients with EBVaGC (P < .001). This finding supports the concept that EBVaGCs have fewer genetic and epigenetic alterations than EBVnGCs. Expression of major histocompatibility complex class II genes and genes that regulate chemokine activity were more often deregulated in EBVaGCs compared with nontumor tissues. In culture, more T cells migrated to EBV-infected gastric cancer cells than to uninfected cells; migration was blocked with a neutralizing antibody against CXCR3 (a receptor for many chemokines). CONCLUSIONS: Fewer genes are deregulated in EBVaGC than in EBVnGC. Most changes in EBVaGCs occur in immune response genes. These changes might allow EBVaGC to recruit reactive immune cells; this might contribute to the better outcomes of these patients compared with those with EBVnGC.

Recurrent fusion transcripts detected by whole-transcriptome sequencing of 120 primary breast cancer samples.

Relatively few recurrent gene fusion events have been associated with breast cancer to date. In an effort to uncover novel fusion transcripts, we performed whole-transcriptome sequencing of 120 fresh-frozen primary breast cancer samples and five adjacent normal breast tissues using the Illumina HiSeq2000 platform. Three different fusion-detecting tools (deFuse, Chimerascan, and TopHatFusion) were used, and the results were compared. These tools detected 3,831, 6,630 and 516 fusion transcripts (FTs) overall. We primarily focused on the results obtained using the deFuse software. More FTs were identified from HER2 subtype breast cancer samples than from the luminal or triple-negative subtypes (P < 0.05). Seventy fusion candidates were selected for validation, and 32 (45.7%) were confirmed by RT-PCR and Sanger sequencing. Of the validated fusions, six were recurrent (found in 2 or more samples), three were in-frame (PRDX1-AKR1A1, TACSTD2-OMA1, and C2CD2-TFF1) and three were off-frame (CEACAM7-CEACAM6, CYP4X1-CYP4Z2P, and EEF1DP3-FRY). Notably, the novel read-through fusion, EEF1DP3-FRY, was identified and validated in 6.7% (8/120) of the breast cancer samples. This off-frame fusion results in early truncation of the FRY gene, which plays a key role in the structural integrity during mitosis. Three previously reported fusions, PPP1R1B-STARD3, MFGE8-HAPL, and ETV6-NTRK3, were detected in 8.3, 3.3, and 0.8% of the 120 samples, respectively, by both deFuse and Chimerascan. The recently reported MAGI3-AKT3 fusion was not detected in our analysis. Although future work will be needed to examine the biological significance of our new findings, we identified a number of novel fusions and confirmed some previously reported fusions.

The full-length DNA sequence of Epstein Barr virus from a human gastric carcinoma cell line, SNU-719.

The consistent presence of Epstein-Barr virus (EBV) in malignant cells of EBV-associated gastric carcinoma (EBVaGC) suggests it plays an important role during the development of EBVaGC. However, the entire genomic sequence of EBV from EBVaGC has yet to be determined. This study first determined, annotated, and analyzed the full genomic sequence of EBV from the naturally infected gastric carcinoma cell line SNU-719 using next-generation sequencing and comparative analyses. In consistent with the notion that EBV sequence isolates better reflect their geographic area than tissue origin, the SNU-719 EBV (named as GC1) was categorized as an East Asian type I EBV. Compared with the prototype B95.8 sequence, SNU-719 EBV contained 1372 variations, with 937 and 435 within coding and non-coding regions, respectively. Of the 937 variations, 465 were non-synonymous changes, while 472 synonymous changes included partial internal deletions in the coding regions of LMP1 and gp350. The RNAseq transcriptome revealed that multiple BART transcripts comprised the majority of EBV RNA reads. The SNU-719 EBV expressed high levels of BART, LF3, BHLF1, and BNLF2. Evidence of RNA editing at multiple sites in the host chromosome was found; however, no evidence of genome integration was seen. The annotated SNU-719 EBV sequence will be a useful reference in future EBVaGC studies.

Genetic association of APOA5 and APOE with metabolic syndrome and their interaction with health-related behavior in Korean men.

BACKGROUND: Genome-wide association studies have been used extensively to identify genetic variants linked to metabolic syndrome (MetS), but most of them have been conducted in non-Asian populations. This study aimed to evaluate the association between MetS and previously studied single nucleotide polymorphisms (SNPs), and their interaction with health-related behavior in Korean men. METHODS: Seventeen SNPs were genotyped and their association with MetS and its components was tested in 1193 men who enrolled in the study at Seoul National University Hospital. RESULTS: We found that rs662799 near APOA5 and rs769450 in APOE had significant association with MetS and its components. The SNP rs662799 was associated with increased risk of MetS, elevated triglyceride (TG) and low levels of high-density lipoprotein, while rs769450 was associated with a decreased risk of TG. The SNPs showed interactions between alcohol drinking and physical activity, and TG levels in Korean men. CONCLUSIONS: We have identified the genetic association and environmental interaction for MetS in Korean men. These results suggest that a strategy of prevention and treatment should be tailored to personal genotype and the population.

Image-based deep learning model using DNA methylation data predicts the origin of cancer of unknown primary.

Cancer of unknown primary (CUP) is a rare type of metastatic cancer in which the origin of the tumor is unknown. Since the treatment strategy for patients with metastatic tumors depends on knowing the primary site, accurate identification of the origin site is important. Here, we developed an image-based deep-learning model that utilizes a vision transformer algorithm for predicting the origin of CUP. Using DNA methylation dataset of 8,233 primary tumors from The Cancer Genome Atlas (TCGA), we categorized 29 cancer types into 18 organ classes and extracted 2,312 differentially methylated CpG sites (DMCs) from non-squamous cancer group and 420 DMCs from squamous cell cancer group. Using these DMCs, we created organ-specific DNA methylation images and used them for model training and testing. Model performance was evaluated using 394 metastatic cancer samples from TCGA (TCGA-meta) and 995 samples (693 primary and 302 metastatic cancers) obtained from 20 independent external studies. We identified that the DNA methylation image reveals a distinct pattern based on the origin of cancer. Our model achieved an overall accuracy of 96.95 % in the TCGA-meta dataset. In the external validation datasets, our classifier achieved overall accuracies of 96.39 % and 94.37 % in primary and metastatic tumors, respectively. Especially, the overall accuracies for both primary and metastatic samples of non-squamous cell cancer were exceptionally high, with 96.79 % and 96.85 %, respectively.

Synergistic Integration of Machine Learning with Microstructure/Composition-Designed SnO2 and WO3 Breath Sensors.

A high-performance semiconductor metal oxide gas sensing strategy is proposed for efficient sensor-based disease prediction by integrating a machine learning methodology with complementary sensor arrays composed of SnO2- and WO3-based sensors. The six sensors, including SnO2- and WO3-based sensors and neural network algorithms, were used to measure gas mixtures. The six constituent sensors were subjected to acetone and hydrogen environments to monitor the effect of diet and/or irritable bowel syndrome (IBS) under the interference of ethanol. The SnO2- and WO3-based sensors suffer from poor discrimination ability if sensors (a single sensor or multiple sensors) within the same group (SnO2- or WO3-based) are separately applied, even when deep learning is applied to enhance the sensing operation. However, hybrid integration is proven to be effective in discerning acetone from hydrogen even in a two-sensor configuration through the synergistic contribution of supervised learning, i.e., neural network approaches involving deep neural networks (DNNs) and convolutional neural networks (CNNs). DNN-based numeric data and CNN-based image data can be exploited for discriminating acetone and hydrogen, with the aim of predicting the status of an exercise-driven diet and IBS. The ramifications of the proposed hybrid sensor combinations and machine learning for the high-performance breath sensor domain are discussed.

Rational design of hybrid sensor arrays combined synergistically with machine learning for rapid response to a hazardous gas leak environment in chemical plants.

Combinations of semiconductor metal oxide (SMO) sensors, electrochemical (EC) sensors, and photoionization detection (PID) sensors were used to discriminate chemical hazards on the basis of machine learning. Sensing data inputs were exploited in the form of either numerical or image data formats, and the classification of chemical hazards with high accuracy was achieved in both cases. Even a small amount of gas sensing or purging data (input for ∼30 s) input can be exploited in machine-learning-based gas discrimination. SMO sensors exhibit high performance even in a single-sensor mode, presumably because of the intrinsic cross-sensitivity of metal oxides, which is otherwise considered a major disadvantage of SMO sensors. EC sensors were enhanced through synergistic integration of sensor combinations with machine learning. For precision detection of multiple target analytes, a minimum number of sensors can be proposed for gas detection/discrimination by combining sensors with dissimilar operating principles. The Type I hybrid sensor combines one SMO sensor, one EC sensor, and one PID sensor and is used to identify NH3 gas mixed with sulfur compounds in simulations of NH3 gas leak accidents in chemical plants. The portable remote sensing module made with a Type I hybrid sensor and LTE module can identify mixed NH3 gas with a detection time of 60 s, demonstrating the potential of the proposed system to quickly respond to hazardous gas leak accidents and prevent additional damage to the environment.

Central neurocytoma exhibits radial glial cell signatures with FGFR3 hypomethylation and overexpression.

We explored the genomic events underlying central neurocytoma (CN), a rare neoplasm of the central nervous system, via multiomics approaches, including whole-exome sequencing, bulk and single-nuclei RNA sequencing, and methylation sequencing. We identified FGFR3 hypomethylation leading to FGFR3 overexpression as a major event in the ontogeny of CN that affects crucial downstream events, such as aberrant PI3K-AKT activity and neuronal development pathways. Furthermore, we found similarities between CN and radial glial cells based on analyses of gene markers and CN tumor cells and postulate that CN tumorigenesis is due to dysregulation of radial glial cell differentiation into neurons. Our data demonstrate the potential role of FGFR3 as one of the leading drivers of tumorigenesis in CN.

Direct oxidation growth of P-type semiconducting CuO nanowires.

P-type copper oxide nanowires (NWs) were grown on metallic copper plates and sapphire substrates. Significant variations in the morphology and distribution of the NWs, due to underlying differences in the growth mechanism and the NW densities, were observed based on the nature of the substrate utilized. The use of copper plates induced an extremely high density of copper oxide nanowires on temperature-dependent copper oxide layers. However, the sapphire substrates gave rise to highly superior CuO NWs without any involvement of an oxide layer, leading to a low density of copper oxide NWs. Systematic characterization of the as-grown copper oxide NWs using X-ray photoelectron microscopy and Raman spectroscopy indicated that the NWs were comprised of CuO with Cu2+ metallic ions.

Extraction of quantitative parameters for describing the microstructure of solid oxide fuel cells.

Digital quantification of a two-dimensional structure was applied to a GDC(Gd2O3-doped CeO2)/LSM(La0.85Sr0.15MnO3) composite cathode employed for solid oxide fuel cells. With the aid of high-resolution imaging capability based on secondary and backscattered electron images, two-dimensional electron micrographs were converted to digital binary files using an image processing tool combined with the line intercept method. Statistical analysis combined with a metallurgical tool was employed to determine microstructural factors, i.e., volume fraction, size distribution, and interconnectivity. The current work reports the quantification of the two-dimensional structural images of GDC/LSM composites applicable to solid oxide fuel cells, with the aim of obtaining the volume fraction, size distribution, and interconnectivity as functions of composite composition. The volume fractions of the solid constituent phases exhibit compositional dependence in cathodes; however, LSM interconnectivity increases gradually as a function of LSM composition, whereas that of GDC decreases significantly at 50 wt% LSM.

Antimicrobial activities of Asian plant extracts against pathogenic and spoilage bacteria.

This study was conducted to investigate the antimicrobial effects of 300 Asian plant extracts (PEs) against pathogenic and spoilage bacteria. The antimicrobial activities were examined using agar well or agar disc diffusion, and micro-titer methods. Results revealed that PEs exhibited higher antimicrobial effects against Gram-positive bacteria compared than against Gram-negative bacteria. With few exceptions, PEs delayed the lag time (LT) of pathogenic bacteria (1.17-3.75 times). Among PEs tested, Alchornea trewioides (AT) and Erodium stephanianum (ES) were the most effective in inhibiting pathogenic and spoilage bacteria. In the study evaluating the effect on the growth inhibition in the broth, Acetobacter aceti was inhibited at 2.77 and 3.02 log CFU/mL by the combination treatment of AT+nisin and ES+nisin after storage for 7 days, respectively. Although further investigations are needed to clarify the antimicrobial mechanism of PEs, this study demonstrated that antimicrobial efficacy varied with PE types, solvents, and bacteria. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01182-0.

Molecular characterization of human respiratory syncytial virus in Seoul, South Korea, during 10 consecutive years, 2010-2019.

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections and hospitalization in infants and young children. Here, we analyzed the genetic diversity of RSV using partial G gene sequences in 84 RSV-A and 78 RSV- B positive samples collected in Seoul, South Korea, for 10 consecutive years, from 2010 to 2019. Our phylogenetic analysis revealed that RSV-A strains were classified into either the ON1 (80.9%) or NA1 (19.0%) genotypes. On the other hand, RSV-B strains demonstrated diversified clusters within the BA genotype. Notably, some sequences designated as BA-SE, BA-SE1, and BA-DIS did not cluster with previously identified BA genotypes in the phylogenetic trees. Despite this, they did not meet the criteria for the assignment of a new genotype based on recent classification methods. Selection pressure analysis identified three positive selection sites (amino acid positions 273, 274, and 298) in RSV-A, and one possible positive selection site (amino acid position 296) in RSV-B, respectively. The mean evolutionary rates of Korean RSV-A from 1999 to 2019 and RSV-B strains from 1991 and 2019 were estimated at 3.51 × 10-3 nucleotides (nt) substitutions/site/year and 3.32 × 10-3 nt substitutions/site/year, respectively. The population dynamics in the Bayesian skyline plot revealed fluctuations corresponding to the emergence of dominant strains, including a switch of the dominant genotype from NA1 to ON1. Our study on time-scaled cumulative evolutionary analysis contributes to a better understanding of RSV epidemiology at the local level in South Korea.

Identification of a Complex Karyotype Signature with Clinical Implications in AML and MDS-EB Using Gene Expression Profiling.

Complex karyotype (CK) is associated with a poor prognosis in both acute myeloid leukemia (AML) and myelodysplastic syndrome with excess blasts (MDS-EB). Transcriptomic analyses have improved our understanding of the disease and risk stratification of myeloid neoplasms; however, CK-specific gene expression signatures have been rarely investigated. In this study, we developed and validated a CK-specific gene expression signature. Differential gene expression analysis between the CK and non-CK groups using data from 348 patients with AML and MDS-EB from four cohorts revealed enrichment of the downregulated genes localized on chromosome 5q or 7q, suggesting that haploinsufficiency due to the deletion of these chromosomes possibly underlies CK pathogenesis. We built a robust transcriptional model for CK prediction using LASSO regression for gene subset selection and validated it using the leave-one-out cross-validation method for fitting the logistic regression model. We established a 10-gene CK signature (CKS) predictive of CK with high predictive accuracy (accuracy 94.22%; AUC 0.977). CKS was significantly associated with shorter overall survival in three independent cohorts, and was comparable to that of previously established risk stratification models for AML. Furthermore, we explored of therapeutic targets among the genes comprising CKS and identified the dysregulated expression of superoxide dismutase 1 (SOD1) gene, which is potentially amenable to SOD1 inhibitors.

PCW-A1001, AI-assisted de novo design approach to design a selective inhibitor for FLT-3(D835Y) in acute myeloid leukemia.

Treating acute myeloid leukemia (AML) by targeting FMS-like tyrosine kinase 3 (FLT-3) is considered an effective treatment strategy. By using AI-assisted hit optimization, we discovered a novel and highly selective compound with desired drug-like properties with which to target the FLT-3 (D835Y) mutant. In the current study, we applied an AI-assisted de novo design approach to identify a novel inhibitor of FLT-3 (D835Y). A recurrent neural network containing long short-term memory cells (LSTM) was implemented to generate potential candidates related to our in-house hit compound (PCW-1001). Approximately 10,416 hits were generated from 20 epochs, and the generated hits were further filtered using various toxicity and synthetic feasibility filters. Based on the docking and free energy ranking, the top compound was selected for synthesis and screening. Of these three compounds, PCW-A1001 proved to be highly selective for the FLT-3 (D835Y) mutant, with an IC50 of 764 nM, whereas the IC50 of FLT-3 WT was 2.54 µM.

Charge-trapping characteristics of Al2O3/Cu/Al2O3 nanolaminate structures prepared through atomic layer deposition.

The nanolaminate Al2O3/Cu/Al2O3 structures were constructed on p-type Si (001) substrates using atomic layer deposition (ALD) process with the aim to fabricating nonvolatile charge-trap memories. Low temperature Cu thin layers were deposited through plasma-enhanced atomic layre depositon of Cu aminoalkoxide (Cu(dmamb)2) combined with hydrogen plasma and Al2O3 layers were prepared by thermal atomic layer deposition of trimethylaluminum (TMA) combined with H2O. Nonvolatile features were confirmed using capacitance-voltage (C-V) measurements. The copper film functions as a charge-trapping layer and the Al2O3 thin layers were employed as tunneling and control oxide layers. Line shapes and binding energies of Cu metal and the thin layer of 6 nm Cu in nanolaminate structures were observed in the X-ray photoelectron spectroscopy (XPS) and high resolution transmission electron microscopy (TEM) image. The V(FB) shift width of the Al2O3 (28 nm)/Cu (6 nm)/Al2O3 (4.2 nm)/Si laminate structure is found to be 4.75 V in voltage sweeping between -10 and +10 V, leading to the trap density of 1.68 x 10(18) cm(-3).