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Treatment efficacy of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is diverse even in non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. Extraordinary long-term responses sustained over 3 years among NSCLC patients treated with afatinib, an EGFR-TKI, have been reported, but how to predict such long-survivors has not been clarified. A multi-institutional prospective observational study, based on comprehensive genomic examination performed with next generation sequencing (NGS) of circulating tumor DNA (ctDNA), was conducted to identify potential predictive markers of long-term response to afatinib. Twenty-nine patients with advanced stage NSCLC and EGFR driver mutations detected by standard techniques were enrolled. ctDNA from plasma collected before afatinib treatment was analyzed by Guardant360®. ctDNA was detected in 25 of the 29 samples. Median progression-free survival was shorter in patients whose tumors having EGFR copy number gain (7.0 vs 23.0 months, p = 0.022). The impact of EGFR copy number on cell proliferation and the antitumor effect of afatinib were evaluated using genome-editing lung cancer cell lines. HCC827 with EGFR amplification were relatively resistant to afatinib at concentrations below 0.5 nM, but genome-edited derivatives of HCC827 with decreased EGFR copy number demonstrated growth inhibition with 0.1 nM afatinib. The absence of EGFR copy number gain detected in ctDNA may be predictive markers of long-term response to afatinib. Comprehensive genomic analysis could lead to more accurate prediction of EGFR-TKI efficacy.

Purpose Dealing with variants of unknown significance (VUS) is an important issue in the clinical application of NGS-based cancer gene panel tests. We detected a novel ERBB2 extracellular domain VUS, c.1157A > G p.(E401G), in a cancer gene panel test. Since the mechanisms of activation by ERBB2 extracellular domain (ECD) variants are not fully understood, we aimed to clarify those mechanisms and the biological functions of ERBB2 E401G. Methods ERBB2 E401G was selected as VUS for analysis because multiple software tools predicted its pathogenicity. We prepared ERBB2 expression vectors with the E401G variant as well as vectors with S310F and E321G, which are known to be activating mutations. On the basis of wild-type ERBB2 or mutant ERBB2 expression in cell lines without ERBB2 amplification or variants, we evaluated the phosphorylation of human epidermal growth factor receptor 2 and related proteins, and investigated with molecular dynamics (MD) simulation the mechanisms conferred by the variants. The biological effects of ERBB2 E401G were also investigated, both in vitro and in vivo. Results We found that ERBB2 E401G enhances C-terminal phosphorylation in a way similar to S310F. MD simulation analysis revealed that these variants maintain the stability of the EGFR-HER2 heterodimer in a ligand-independent manner. Moreover, ERBB2 E401G-transduced cells showed an increased invasive capacity in vitro and an increased tumor growth capacity in vivo. Conclusion Our results provide important information on the activating mechanisms of ERBB2 extracellular domain (ECD) variants and illustrate a model workflow integrating wet and dry bench processes for the analysis of VUS detected with cancer gene panel tests.

Liquid biopsy with circulating free DNA (cfDNA) is a recommended alternative method of re-biopsy. Quality control with cfDNA is indispensable for precise examinations, and it is desirable to achieve high-quality cfDNA separation. We investigated two issues: the influence of pre-analytical procedures on cfDNA analysis performed as a routine procedure in a standard clinical laboratory, and the extent of deterioration of cfDNA quality due to long-term storage. Comparisons among blood collection tube types, storage temperatures, and periods of blood separation were performed in terms of cfDNA quantification, cfDNA size distribution, and detection of EGFR mutations. Quality of cfDNA was better with collection tubes containing 3.2% sodium citrate than with those containing EDTA 2K, and was maintained with storage at 4° C for up to 72 h after blood collection, equivalent to results with cell-stabilizing blood collection tubes. Analysis of cfDNA stored for 7 years showed that samples with low allele frequency (AF) deteriorated more readily than samples with high AF. Despite the same storage period and extraction method, AF of plasma stored for 7 years was remarkably lower than that of cfDNA. However, deterioration due to long-term plasma storage was overcome by changing the DNA extraction method from a silica membrane spin column to a cellulose magnetic beads system. These results can guide the establishment of standardized pre-analytical procedures for liquid biopsy with cfDNA.

The clinical utility of plasma DNA for detecting cancer-specific mutations has rapidly achieved recognition, but reliability has not been established because of relatively low mutation-detection rates compared with those from tissue re-biopsy. To address this shortcoming we examined efficiency, in terms of mutation detection, of an automated DNA extraction system that uses cellulose magnetic beads. A fully automated, highly sensitive point-mutation-detection method, mutation-biased PCR and quenching probe (MBP-QP) system, was used for this study. Plasma DNA was extracted from 61 plasma samples collected from patients with advanced non-small cell lung cancer. Extraction was performed manually with 200 μl plasma (200-M) by using a silica membrane spin column system or an automated system using 200 μl (200-A) or 1000 μl (1000-A) plasma. Median DNA yield quantified by real-time PCR was 4.4, 4.5, and 17.3 ng with the three methods, respectively. Sensitivity for detecting epidermal growth factor receptor (EGFR) L858R point mutation was 36.6%, 58.5%, and 77.5%, and specificity was 93.3%, 100%, and 96.7%, respectively. Concordance rates were 60.6%, 76.1%, and 85.7%. The size distribution of plasma DNA with automated extraction was bimodal with modes at about 170 bp and 5 Kb, and plasma DNA of both sizes included tumor-derived DNA. In this report, we demonstrate that automated DNA extraction using cellulose magnetic beads can improve mutation-detection rates with plasma DNA in association with two overall sizes of DNA fragments recovered by this DNA isolation system. Examining the biological characteristics of these fragments will be the subject of further investigation.

In this work, the mechanical behavior of as-received and hydrogenated Zircaloy-4 fuel claddings was investigated by the newly developed advanced expansion due to compression (A-EDC) test and the conventional uniaxial tension (UT) test at room temperature, in order to, respectively, understand the hydride-induced embrittlement in tube longitudinal and hoop directions. The UT experimental results showed that the mechanical strength in the longitudinal direction slightly increased with hydrogen content, whereas the maximum strain decreased greatly with hydrogen increasing. In the case of A-EDC tests, the mechanical performance in the hoop direction seemed insensitive to the hydrogen content; no obvious decline in maximum strain was observed until 800 ppm H. The comparison between these two tests clearly reveals that the hydride-induced embrittlement is preferential to occur in the longitudinal direction, compared with the sluggish response in the hoop direction, which implied the enhanced ductility a...

Pemetrexed is a key anticancer agent for treatment of advanced non-small cell lung cancer (NSCLC). Pemetrexed is generally well tolerated, but individual-patient differences exist in severity of adverse events. Our study aimed to characterize the adverse events of pemetrexed that result in discontinuation of chemotherapy and to identify risk factors associated with those adverse events. We retrospectively studied the incidence of adverse events in 257 patients with NSCLC who received pemetrexed (P) with or without bevacizumab (B) and/or carboplatin (C): P, PB, CP, or CPB. Patients whose chemotherapy was discontinued were divided into two groups according to adverse events and disease progression. Grade 2/3 nausea, fatigue with P and PB, and rash with CP and CPB occurred more frequent in the adverse events group than in the disease progression group. Multivariate analysis indicated that grade 2/3 nausea [odds ratio (OR) 9.94; 95% confidence interval (CI) 1.46-67.37; p = 0.01] and fatigue (OR 10.62; CI 1.60-70.20; p = 0.01) with P or PB, and rash (OR 6.12; CI 1.34-27.88; p = 0.01) with CP or CPB, were independent risk factors for discontinuation of chemotherapy. Administration of dexamethasone at doses less than 4 mg after the day of pemetrexed administration was associated with nausea following P or PB (OR 11.08; 95% CI 1.02-119.95; p = 0.04). Grade 2/3 nausea and fatigue with P or PB, and rash with CP or CPB, were associated with discontinuation of chemotherapy.

We have prepared tetraazanaphthacene-based liquid crystal 1 having two racemic alkoxy chains. Compound 1 self-organizes to form the rectangular columnar liquid-crystalline phase over a wide temperature range including room temperature. However, compound 2 having two linear alkoxy chains is incapable of self-organizing superstructures. Since cyclic voltammetry revealed that 1 has two-step and two-electron reductions, 1 is expected to function as electron-transporting material.

The preparation of two liquid crystals composed of a redox-active tetraazanaphthacene (TANC) framework is reported. The materials form smectic A (SmA) thin-film liquid-crystalline (LC) phases over a wide temperature range. Cyclic voltammetry analysis revealed that LC TANCs behave as organic electron acceptors. The electron mobilities of the thin films were determined by time- of-flight (TOF) measurements, which are the order of 10(-4) cm(2) V(-1) s(-1) in the SmA LC phase. This value is two orders of magnitude larger than those of amorphous organic semiconductors. To the best of our knowledge, very few reports exist on the electron-transporting behaviors of LC N-heteroacene semiconductors.

Zr–Si alloys were designed to contain eutectic phase surrounding the parent phase to suppress creep behavior of claddings. Creep tests conducted at 294–573 K showed that creep behavior was inhibited and that the creep failure time of new Zr alloy became longer than that of a conventional alloy: Zircaloy-4. Results show that the eutectic phase can suppress creep at operating temperatures prevailing in current nuclear power plants.

Objectives Discoidin domain receptor (DDR) 2 mutations have recently been reported to be candidate targets of molecular therapy in lung squamous cell carcinoma (SQCC). However, the status of DDR2 expression and mutations, as well as their precise roles in lung SQCC, have not been clarified. We here report DDR2 mutation and expression status in clinical samples and its role of lung SQCC. Materials and methods We investigated DDR2 expression and mutation status in 44 human clinical samples and 7 cell lines. Biological functions of DDR2 were assessed by in vitro cell invasion assay and animal model experiments. Results Endogenous DDR2 protein expression levels were high in one cell line, PC-1, and immunohistochemistry of lung cancer tissue array showed high levels of DDR2 protein in 29% of lung SQCC patients. A mutation (T681I) identified in lung SQCC and the cell line EBC-1 was detected among 44 primary lung SQCC samples and 7 lung SQCC cell lines. Although Forced expression of DDR2 and its mutant (T681I) led to induce SQCC cell invasion in vitro, only wild type DDR2 enhanced lung metastasis in an animal model. We also found that ectopic expression of DDR2 induced MMP-1 mRNA expression accompanied by phosphorylation of c-Jun after treatment with its ligand, collagen type I, but DDR2 with the T681I mutation did not, suggesting that T681I mutation is an inactivating mutation. Conclusion Overexpression of DDR2 might contribute to tumor progression in lung SQCC. The overexpression of DDR2 could be potential molecular target of lung SQCC.

A patient with cancer of unknown primary underwent an NGS-based multiplex gene assays (OncoPrime). As a result, a novel variant of HER2 (E401G) which is a missense alteration located in the extracellular domain of the HER2 protein was detected. This alteration has not been reported (COSMIC, Nov 2018) and its effect on protein function is unknown. Sanger sequencing using genomic DNA from peripheral blood sample showed that the variant was a somatic variant. HER2 E401G variant was confirmed by Sanger sequencing using genomic DNA extracted from the formalin-fixed paraffin-enbedded tumor tissues. In spite of somatic variant, the variant allele frequency was relatively high (55.8%). This result suggested the amplification of HER2 gene, FISH analysis confirmed the copy number gain of HER2 gene (21 copies). The prediction tools in silico (SIFT, PolyPhen-2 and PROVEAN) suggested pathogenicity of the variant, so we are now performing the functional analysis of the mutant HER2 protein. Many variants of unknown significance have been identified through clinical sequencing, establishing a database of integrated information of variant is essential to implement precision medicine of oncology. This study was approved by the Ethics Committee and the patient provided written informed consent for the use of genomic and clinical data for research purposes. Citation Format: Yohei Harada, Tomomi Kashiwada, Akemi Sato, Tomonori Abe, Chiho nakashima, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane. Analysis of a novel variant of HER2 detected by clinical sequencing in cancer of unknown primary [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 855.

Liquid biopsy using circulating tumor DNA (ctDNA) has been spread world-wide. We established fully automatic sensitive mutation detection system, mutation-biased PCR and quenched probe system (MBP-QP) method, and accomplished multicenter prospective study to investigate the utility of ctDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). The results of the clinical study showed that ctDNA was frequently detected in lung cancer patients with distant metastasis, and detection of ctDNA was associated with poor prognosis. To investigate the significance of these clinical data, we analyzed biological and clinical characteristics of ctDNA. We examined 130 plasma samples from 92 lung cancer patients in addition to 18 benign pulmonary disease patients and 20 healthy individuals at Saga University Hospital.Circulating free DNA (cfDNA) was extracted from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. The DNA concentration was quantified by Quantus®, the fluorescent measurement of dsDNA intercalated dye, and the DNA size distribution was analyzed by Bioanalyzer®, capillary electrophoresis system. We found difference of cfDNA size distribution between lung cancer patients and healthy individuals: former showed two peaks of 5kb and 170bp, and latter showed single peak of 170bp. The DNA concentration was higher in lung cancer patients compared to those in benign pulmonary disease patients and healthy individuals. Among lung cancer patients, DNA concentration was increased in those with advanced stages, especially in presence of metastasis. In addition, 5 kb fragments DNA was significantly increased in these cases compared to 170 bp fragments DNA. To investigate which fragment contained tumor-derived DNA, 170 bp and 5 kb fragments were separately isolated, and EGFR mutation, L858R was examined. L858R was detected in both ctDNA fragments, 170 bp and 5 kb, indicating that both sized DNA fragments contain tumor-derived DNA. In order to evaluate DNA resolution, we measured the concentrations of DNase1 and DNase1L3 in plasma, but no significant difference was observed among three groups. Although the 170 bp fragments DNA are well known as an apoptotic product, the origin of 5 kb long fragments DNA has not been clarified. We hypothesized that long fragment DNA has a mechanism to escape from DNase, extracellular vesicles or DNA associated protein. In order to examine that, We analyze whether long fragment DNA is associated with extracellular vesicles or DNA associated protein. Citation Format: Tomonori Abe, Chiho Nakashima, Akemi Sato, Yohei Harada, Eisaburo Sueoka, Shinya Kimura, Naoko Aragane. Characteristics of circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 420.

Herein, we report the syntheses and characterization of liquid-crystalline (LC) 5,6,13,14-tetraazapentacene (BTANC) derivatives. A LC BTANC derivative self-organized to form a layered smectic A LC ...

Liquid biopsy using circulating tumor DNA (ctDNA) has been spread world-wide. We established fully automatic sensitive mutation detection system, mutation-biased PCR and quenched probe system (MBP-QP) method, and accomplished multicenter prospective study to investigate the utility of ctDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI). The results of the clinical study showed that ctDNA was frequently detected in lung cancer patients with distant metastasis, and detection of ctDNA was associated with poor prognosis. In metastatic animal model using immunodeficient mice, NOJ mice, the amount or ctDNA was associated with tumor progression such as tumor burden and metastasis. Based on these evidences, we hypothesized that ctDNA has some biological effects on tumor progression. In order to examine that, we analyzed biological and clinical characteristics of ctDNA. ctDNA was extracted from 1000μl plasma by automated DNA extraction system using cellulose magnetic beads. Compared to circulating free DNA (cfDNA) isolated from healthy volunteers, size distribution of ctDNA or cfDNA using a capillary electrophoresis system was different, that is one peak around the size of 170 bp in healthy individuals, and two peaks, 170 bp and 5 kb in advanced lung cancer patients. We next examined 130 plasma samples from 92 lung cancer patients in addition to 18 benign pulmonary disease patients and 20 healthy individuals at Saga University Hospital. The DNA concentration quantified by Quantus®, the fluorescent measurement of dsDNA intercalated dye, was higher in lung cancer patients compared to those in benign pulmonary disease patients and healthy individuals. Among lung cancer patients, DNA concentration was increased in those with advanced stages, especially in presence of metastasis. In addition, 5 kb fragments were significantly increased in these cases compared to 170 bp fragments. To investigate which fragment contained tumor-derived DNA, 170 bp and 5 kb fragments were separately isolated, and EGFR mutation, L858R was examined. L858R was detected in both ctDNA fragments, 170 bp and 5 kb, indicating that both sized DNA fragments contain tumor-derived DNA. Although the 170 bp short fragments of ctDNA are well known as an apoptotic product, the origin of 5 kb long fragments has not been clarified. We have examined whole ctDNA sequence using next generation sequencing, and compared two fragments. In addition, origins of each fragment have been investigated, and biological effects on tumor progression will be analyzed. Citation Format: Tomonori Abe, Chiho Nakashima, Akemi Sato, Eisaburo Sueoka, Shinya Kimura, Naoko Aragane. Characteristics of circulating tumor DNA in lung cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3408.

The usefulness of circulating free DNA (cfDNA) for analysis of genetic alterations is largely accepted. We accomplished multicenter prospective study to investigate sequential change of cfDNA in lung cancer patients who acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. The detection frequency was elevated as cancer progressed, and the prognosis of the patients in which cfDNA was detected was poorer than those not detected. These results suggest that appearance of cfDNA was associated with tumor progression. To verify that, animal experiment using immunodeficient mice, NOJ mice, was performed. After transplantation with human lung cancer cell line, H1975 carrying EGFR L858R, and T790M, into dorsal flanks of these mice, systemic metastasis occurred. cfDNA was sequentially analyzed, resulting that the amount of cfDNA was correlated with tumor burden and metastatic status. In spite of these results, origin, kinetics or possible function on tumor progression of cfDNA has not been elucidated. We have reported there were large sized DNA fragments, around 5 Kb, which is longer than 170 bp of cfDNA conventionally detected in peripheral blood of advanced cancer patients. Exosomes, extracellular vesicles detected in peripheral blood has been reported to be involved in tumor progression through vesicle-mediated communication. In general, exosomes deliver protein, lipid and RNA, and few DNA was contained. However, some researchers reported a large proportion of cfDNA was localized in exosomes. To investigate the origin and localization of cfDNA in peripheral blood, we analyzed relationship between cfDNA and exosomal DNA. We isolated both of cfDNA and exosomal DNA simultaneously, from plasma of healthy individuals and advanced non-small cell lung cancer patients, and compared the DNA yield, DNA size distribution and EGFR mutation detection rate. Localization of DNA in exosome is also investigated using fluorescent dye. We used Total Exosome Isolation Kit® from plasma with/without proteinase K for isolation of exosome from 200 uL plasma, and extracted DNA using Maxwell RSC® circulating cell free DNA cartridge. The proportion of exosomal DNA /cfDNA (e/c DNA ratio) varied individually, but e/c DNA ratio was lower in plasma from cancer patients than healthy individuals. However, large sized fragments of cfDNA were observed in exosomal DNA in lung cancer patients. These results suggest that exosomal DNA might have some interaction with large sized cfDNA fragments observed in plasma isolated from advanced cancer patients. Citation Format: Chiho Nakashima, Tomonori Abe, Akemi Sato, Tomomi Nakamura, Kazutoshi Komiya, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane. Investigation of origin of circulating free DNA: Is exosomal DNA the carrier [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3646.

River cross sectional form had settled in the dynamic equilibrium form at the relationship between discharge, the river bed slope and bed material when river improvement is not progressing so much. But, river channels do not seem to satisfy the equilibrium condition of sediment transport due to river improvement work in progress. As the result, many problems have arisen on river management. Sediment transport became discontinuous by the dam and removing large size of bed materials as stones and gravel from rivers is mentioned. In this study, we describe the bed stabilization mechanism of the stony bed rivers found by the field experiment of the Joganji River and the difference in sediment transport mechanism of the stony bed river and the gravel bed river. Moreover, we insist on the problem on washing out of dam reservoir sediment and the necessity for the new bed variation analysis method in the stony bed rivers.

16 pages, 4 figures, 6 tables., Water samples were collected from rivers and estuarine environments within the Florida Coastal Everglades (FCE) ecosystem, USA, and ultrafiltered dissolved organic matter (UDOM; 1 kDa) was isolated for characterization of its source, bioavailability and diagenetic state. A combination of techniques, including 15N cross-polarization magic angle spinning nuclear magnetic resonance (15N CPMAS NMR) and X-ray photoelectron spectroscopy (XPS), were used to analyze the N components of UDOM. The concentrations and compositions of total hydrolysable amino acids (HAAs) were analyzed to estimate UDOM bioavailability and diagenetic state. Optical properties (UV–visible and fluorescence) and the stable isotope ratios of C and N were measured to assess the source and dynamics of UDOM. Spectroscopic analyses consistently showed that the major N species of UDOM are in amide form, but significant contributions of aromatic-N were also observed. XPS showed a very high pyridinic-N concentration in the FCE–UDOM (21.7 ± 2.7%) compared with those in other environments. The sources of this aromatic-N are unclear, but could include soot and charred materials from wild fires. Relatively high total HAA concentrations (4 ± 2% UDOC or 27 ± 4% UDON) are indicative of bioavailable components, and HAA compositions suggest FCE–UDOM has not undergone extensive diagenetic processing. These observations can be attributed to the low microbial activity and a continuous supply of fresh UDOM in this oligotrophic ecosystem. Marsh plants appear to be the dominant source of UDOM in freshwater regions of the FCE, whereas seagrasses and algae are the dominant sources of UDOM in Florida Bay. This study demonstrates the utility of a multi-technique and multi-proxy approach to advance our understanding of DON biogeochemistry., This study was funded by the National Oceanographic and Atmospheric Administration (COP R.J.) and the National Science Foundation as part of the FCE-LTER program (DEB-9910514).

Variations in the amount and composition of immobilized nitrogen (N) in major soil organic matter fractions were investigated in a 730-day soil incubation experiment using 15N-labeled urea and 15N nuclear magnetic resonance spectroscopy with the cross polarization/magic angle spinning (15N CPMAS NMR) method. After 730 days, 24.7% of the applied N was recovered from the soil as organic N. The urea-derived N recovered from humic acids and humin decreased from 11.2 and 33.8% of the applied amount after 14 days to 1.6 and 20.4% after 730 days, respectively. When these values were corrected for the microbial biomass (MB) N, they ranged from 9.0 to 1.2% and 28 to 18%, respectively. The proportion of urea-derived N recovered from fulvic acids was low, ranging between 0.4 and 5.8% (with MB N) or 5.6% (without MB N) of the applied amount, whereas that from water-soluble nonhumic substances (WS-NHS; NHS in the fulvic acid fraction) remained high, 28–33% of the applied amount after correction for the contribution of MB N up to day 365, and decreased to 0.9% thereafter. The 15N CPMAS NMR spectra of humic acids, fulvic acids, and humin showed the largest signal at −254 to −264 ppm, corresponding to peptide/amide N. The proportions of heterocyclic, peptide/amide, guanidine/aniline, and free amino N in the urea-derived humic acid N were 3–7, 83–90, 5–7, and 2–4%, respectively. More than 80% loss of the urea-derived humic acid N did not markedly alter their composition. No time-dependent variations were also observed for the proportions of respective N functional groups in humin N, which were 3–5, 71–78, 12–17, and 6–10% in the same order as above. These results suggest the greater importance of physical stability than structural variation for the initial accumulation of organic N in soil.

Nitrogen in humic substances has an important function as a reservoir of plant nutrient. To elucidate the stability of humic acid N in soil with respect to chemical structure, X-ray photoelectron spectroscopy (XPS) and 15N cross polarization/magic angle spinning nuclear magnetic resonance (CPMAS NMR) spectroscopy were applied to humic acids with various degrees of humification. The composition of the non-hydrolyzable fraction of humic acid N remaining after step-wise acid hydrolysis was also investigated by XPS. The proportion of non-hydrolyzable N in aromatic N (including imine, heterocyclic C N, and aromatic amine), 26.2 ± 5.4%, was greater than that in peptide bond N or primary amine N, 4.2–21.5%. However, peptide bond N was the dominant species in the non-hydrolyzable humic acid N, 58.7–79.5%, and aromatic N and primary amine N accounted for 12.2–22.3% and 5.6–19.8%, respectively. The proportions of heterocyclic N, peptide/amide N, guanidine/aniline N, and free amino N in total N, estimated by 15N CPMAS NMR, were 5–21%, 59–83%, 4–8%, and 3–12%. The relative concentration of peptide/amide N estimated by 15N CPMAS NMR correlated negatively with the degree of humification that was estimated from two variables indicating the degree of darkening. In general, significant differences between the proportion of peptide/amide N (or peptide bond N) in total N estimated by 15N CPMAS NMR and XPS were not detected. The proportion of heterocyclic N in total N estimated by 15N CPMAS NMR correlated positively with the degree of humification. The absence of significant differences between the proportions of aromatic N (XPS) and heterocyclic N (15N CPMAS NMR) in total N suggested that aromatic amines are minor constituents in humic acid N. Since pyrrolic N is not included in XPS measurements of aromatic N, it was considered that the relative concentration of heterocyclic C N increases with increasing degree of humification.

The composition of nitrogen (N) species in 11 humic acids obtained from a wide variety of soils was investigated by X-ray photoelectron spectroscopy (XPS). The N1s spectra of the humic acids were fitted by three Gaussian curves with binding energy at 399.0 ± 0.1, 400.4 ± 0.1, and 402.4 ± 0.2 eV, whi

Incorporation of newly-immobilized N into major soil organic matter fractions during a cropping period under paddy and upland cropping systems in the tropics was investigated in Jawa paddy fields with and without fish cultivation and a Sumatra cassava field in Indonesia. 15N-labelled urea (15N urea) was applied as basal fertilizer, and the soil samples were collected after harvest. The percentage of distribution of the residual N in soil from 15N urea into the humic acids, fulvic acid fraction, and humin were 13.1–13.9, 19.0–20.5, and 53.4–54.3%, respectively, for the Jawa paddy soils, and 14.9, 27.4, and 52.4%, respectively, for the Sumatra cassava soil. These values were comparable to the reported ones for other climatic zones. The percentage of distribution of 15N urea-derived N into humic acids was larger than that of total N into the same fraction in all the soils. The distribution into the fulvic acid fraction was also larger for 15N urea-derived N than for total N in the Jawa soils. Humic ...

Cassava is the fourth most important staple food crop in Asia and main crop in the cropping systems used in upland areas. Among staple crops, cassava most abundantly absorbs potassium (K), suggesting that a nutrient disorder might occur among the intercrops. A nutrient disorder in intercrops may affect the fate of applied nitrogen (N) through the growth of intercrops. Strong K-absorption ability of cassava can be potentially antagonistic to other crops. If so, when the soil K level is critical in an intercropping system, K application may result in increased yield through mitigation of the K antagonism among intercrops, accompanied with nutrition improvement of other nutrients. We conducted a field experiment to evaluate the growth of crops and the fate of N fertilizer with reference to the application rate of K in a cassava-based cropping system in South Sumatra. The results obtained were as follows: 1) The dry weight values and yield of cassava, upland rice, and corn without K application were ...

信濃川小千谷・越路地区には, 砂礫堆の形成の防止を含む河床の安定化等を目的とした妙見堰が堰上流の河床変動対策工と併せて1990年に完成している. 本研究では, 最初に河床変動対策工の設置位置と堰上流の河床変動との関係, 堰の整流効果が堰下流の河床変動に与える影響について検討を行った. さらに妙見堰完成後の堰上下流の河床変動状況から, 現地観測結果と模型実験結果の対応, 水理解析モデルによる現地観測結果の再現性について検討を行った. その結果, 現地観測, 模型実験, 数値シミュレーションは, 堰を含めた河床変動対策後の河床形状についてほぼ同等の結果を得ることができ, ここで示した模型実験法および数値シミュレーション法は河床変動対策工の設計法として有効な手段となることを明らかにした.

The usefulness of liquid biopsy to detect mutations from cancer patients has been well recognized today. However, because the mutation detection rates from plasma DNA were relatively lower than those of tissue re-biopsy, its clinical utility has not been confirmed yet. As previously we reported, we have developed fully automatic high-sensitive point mutation detecting system named mutation-biased PCR and quenched probe (MBP-QP) system for liquid biopsy. Recently, the importance of pre-analytical procedures for plasma DNA anazysis has been highlighted. In this study, we examined whether the automatic DNA extraction system can improve the mutation detection rate in our MBP-QP system. Sixty-one plasma samples were obtained from advanced non-small cell lung cancer patients, and plasma DNA extraction was performed from 200μl plasma by manually (200-M), and 200μl (200-A), 1000μl (1000-A) plasma by automatically. We used silica membrane spin column system for manual DNA extraction, and magnet beads system for automatic DNA extraction procedure. The median DNA concentrations quantified by quantitative real-time PCR of 200-M, 200-A, 1000-A were 4.92, 6.00, 20.1 ng/mL plasma, respectively. In terms of the epidermal growth factor receptor (EGFR) L858R point mutation detection, the sensitivity of 200-M, 200-A, 1000-A were 36.6%, 58.5%, 77.5%, that of the specificity were 93.3%, 100%, 96.7%, and the concordance rates were 60.6%, 76.1%, 85.7%, respectively. The size distribution of automatically extracted plasma DNA represented two peaks characteristics at 170 bp and 5 kb. In this study, we indicate the automatic DNA extraction can improve mutation detection rates in plasma DNA. Citation Format: Chiho Nakashima, Akemi Sato, Tomonori Abe, Tomomi Nakamura, Kazutoshi Komiya, Eisaburo Sueoka, Shinya Kimura, Naoko Sueoka-Aragane, Junichi Kato, Mitsuharu Hirai. Automatic DNA extraction system can improve the EGFR point mutation detection rate of liquid biopsy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2751. doi:10.1158/1538-7445.AM2017-2751

A 54-year-old man was admitted to Saga University hospital with dyspnea on effort and a sensation of pressure in the chest. Chest CT images showed a low-density mass in the mediastinum surrounding the carina and left hilus, causing narrowing of both the left pulmonary artery and left main bronchus. The pathological findings from a surgical biopsy showed markedly fibrotic tissue with lymphocytes and plasmacytes, and we diagnosed idiopathic fibrosing mediastinitis, stage II. Oral glucocorticoid treatment of 30 mg/day prednisolone reduced the mass and improved the narrowing of the left pulmonary artery and left main bronchus. The patient was given low-dosage glucocorticoids as maintenance treatment. Previous reports indicated that idiopathic fibrosing mediastinitis with severe tissue fibrosis is difficult to control with glucocorticoid monotherapy. Here, we report a case of idiopathic fibrosing mediastinitis that was effectively treated with glucocorticoids.

Purpose Gene polymorphism and genetic mutation are related to the therapeutic effect of many drugs, and a simple genetic test method is preferred. To meet this demand, we have developed a platform for genetic testing called i-densy, which can complete fully automated gene polymorphism analysis from whole blood in approximately 90 minutes, and compared it with the existing test method. Also, recent reports indicate that DNA derived from cancer cells are liberated in blood (it is called “circulating cell-free DNA”). Using the i-densy, we here tested EGFR gene T790M mutation in circulating cell-free DNA from a sample derived from advanced lung cancer patient with EGFR-TKI aquired resistance, and then compared with the existing test method. Methods The i-densy newly developed can perform fully automated gene polymorphism testing from 70μL of whole blood and allows typing of 3 polymorphisms or mutations in a single reaction. Four samples can be tested at once with this instrument, and testing is also possible using oral mucosa or purified nucleic acid instead of whole blood. In addition to the i-densy, circulating cell-free DNA was also tested using Mutation Biased PCR (MBP) method that can preferentially amplify mutation sequence. Results As a result of using the i-densy with whole blood for gene testing on 1236C>T, 3435C>T and 2677 G>(T/A) polymorphism of ABCB1 gene related to toxicity and pharmacokinetics of EGFR-TKI erlotinib, results of 1236C>T and 3435C>T matched with that of Taqman assay method and 2677 G>(T/A) matched with Direct sequencing method for all 20 samples. We also extracted free DNA from peripheral blood plasma of a patient, and tested EGFR T790M mutation using Mutation Biased PCR (MBP) method, PNA-LNA PCR clamp method and Cycleave method. Among 19 patients with acquired resistance to EGFR-TKI, number of patients tested positive for T790M were 10 (53%) using MBP method, 3 (16%) using PNA-LNA PCR method and 5 (26%) using Cycleave method. Also, when using MBP method, there was not a single case with T790M mutation detected among cases with continued therapeutic effect or which EGFR-TKI is not used. Conclusion The i-densy newly developed has been identified to be simple and fast that allows accurate gene polymorphism from whole blood, and also confirmed to be useful in clinical practice. As a result of EGFR gene T790M mutation testing from circulating cell-free DNA using the i-densy and MBP method, appearance ratios of acquired resistance mutation were very close to each other (approximately 50%), indicating the high detection performance compared to other methods. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2089. doi:1538-7445.AM2012-2089

e13561 Background: EGFR tyrosine kinase inhibitors (EGFR-TKIs) are widely used to treat lung adenocarcinomas with EGFR activating mutations. However, half of patients acquire resistance because of ...

Although EGFR tyrosine kinase inhibitors (EGFR-TKIs) have great response to lung adenocarinoma with EGFR activating mutations, acquired resistance eventually developed. The half of these patients has the gatekeeper T790M mutation of EGFR in lung cancer tissue. A non-invasive mutation detection system is desired considering the difficulty in obtaining tissue specimens during disease progression_the majority of lung cancer recurrences occur in distant sites. We report a novel non-invasive monitoring system, MBP-QP (mutation-biased PCR and quenching probe) method, to detect the mutation using plasma DNA. The MBP-QP method is combined with mutated biased PCR and genotyping based on the analysis of a melting curve of probe DNA which is bound to the target mutated site using a fluorescence quenching probe. The detection limit was 2 copies of control plasmid, and 0.2 ng of genomic DNA isolated from a lung caner cell line harboring T790M. 0.3 % mutant plasmid could be detected in the mixture of plasmids inserted with EGFR exon 20 with or without T790M. With this method, T790M mutations were detected in plasma DNA of 50% of patients who acquired resistance, but not in primary EGFR-TKI non-responders, patients responding to treatment, or patients not treated with EGFR-TKI, which is consistent with the clinical course. The non-invasive MBP-QP method enabled us to monitor T790M repeatedly and will be useful for determining appropriate lung cancer treatment strategies in practice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4117. doi:10.1158/1538-7445.AM2011-4117