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Supplementary Figures S1-S2 from Rapid Detection of Epidermal Growth Factor Receptor Mutations in Lung Cancer by the SMart-Amplification Process

Purpose: A positive response to gefitinib in non–small cell lung cancer (NSCLC) has been correlated to mutations in epidermal growth factor receptor (EGFR) gene. Previous reports have been based mainly on diagnostic screening by sequencing. However, sequencing is a time-consuming and complicated procedure, not suitable for routine clinical use.Experimental Design: We have developed rapid, simple, and sensitive mutation detection assays based on the SMart Amplification Process (SMAP) and applied it for analyzing EGFR gene mutations in clinical samples. By using SMAP, we can detect mutations within 30 min including sample preparation. To validate the assay system for potential use in clinical diagnostics, we examined 45 NSCLC patients for EGFR mutations using sequencing and SMAP.Results: The outcomes of the SMAP assay perfectly matched the sequencing results, except in one case where SMAP was able to identify a mutation that was not detected by sequencing. We also evaluated the sensitivity and specificity of SMAP in mutation detection for EGFR. In a serial dilution study, SMAP was able to find a mutation in a sample containing only 0.1% of the mutant allele in a mixture of wild-type genomic DNA. We also could show amplification of mutated DNA with only 30 copies per reaction.Conclusions: The SMAP method offers higher sensitivity and specificity than alternative technologies, while eliminating the need for sequencing to identify mutations in the EGFR gene of NSCLC. It provides a robust and point-of-care accessible approach for a rapid identification of most patients likely to respond to gefitinib.

Supplementary Materials and Methods of plasmids, siRNA, shRNA, transfections, western blot analysis, reagents, luciferase reporter assays, chromatin immunoprecipitation, immunocytochemistry, immunohistochemistry, scratch assays, cell proliferation assays, cell cycle analysis, quantitative real-time PCR, migration and invasion assays, animal experiments, microarray analysis, apoptosis assays, and generation of vemurafenib-resistant cell lines.

Table S1. List of siRNA Screening Library Sequences. Table S2. Identification of PDGFD as a ZIC5 Downstream Factor

Insights into mechanisms of drug resistance could extend the efficacy of cancer therapy. To probe mechanisms in melanoma, we performed siRNA screening of genes that mediate the development of neural crest cells, from which melanocytes are derived. Here, we report the identification of ZIC5 as a mediator of melanoma drug resistance. ZIC5 is a transcriptional suppressor of E-cadherin expressed highly in human melanoma. ZIC5 enhanced melanoma cell proliferation, survival, drug resistance, in vivo growth and metastasis. Microarray analysis revealed that ZIC5 downstream signaling included PDGFD and FAK activation, which contributes to drug resistance by enhancing STAT3 activation. Silencing of ZIC5 or PDGFD enhanced the apoptotic effects of BRAF inhibition and blocked survival of melanoma cells resistant to BRAF inhibitors. Furthermore, inhibition of FAK or STAT3 suppressed expression of ZIC5, which was positively regulated by PDGFD, FAK, and STAT3 in a positive feedback loop. Taken together, our results identify ZIC5 and PDGFD as candidate therapeutic targets to overcome drug resistance in melanoma. Cancer Res; 77(2); 366–77. ©2016 AACR.

A Nonomuraea sp. strain MM565M-173N2 was isolated from deep-sea sediment off the Sanriku coast, and new antibiotics were evaluated against carbapenem-resistant Enterobacteriaceae (CRE), which is a problematic group of bacteria because of their antimicrobial resistance. From 220 l of fermented broth from strain MM565M-173N2, we isolated four new antibiotics by gel filtration and HPLC, designated as sealutomicins A (1.8 mg), B (1.5 mg), C (0.8 mg), and D (0.8 mg). Their structures were determined from MS, NMR, and CD spectra. Sealutomicin A was found to be a new enediyne antibiotic, while sealutomicins B-D were aromatized products from sealutomicin A. Sealutomicin A showed strong antibacterial activity (MIC 0.05-0.2 μg ml-1) against CRE.

Introduction Quantitative measurement of anti-A/-B antibody titers is important during ABO-incompatible living kidney transplantation (ABOi-LKT). Methods We conducted a multi-institutional study to measure the antibody titers using the automated column agglutination technique (auto-CAT) and tube test (TT) method in ABOi-LKT recipients. Statistical analysis was performed to evaluate the two methods. Results We examined 111 samples from 35 ABOi-LKT recipients at four institutions. The correlation coefficient of the two methods was >0.9; the concordance rate and clinically acceptable concordance rate for the IgG titers were 60.4% and 88.3%, respectively. Perioperative status did not influence the statistical significance. Parallel changes were observed in the IgG antibody titers measured using the auto-CAT or TT technique by desensitizing therapy in time-course monitoring. Conclusion Auto-CAT is comparable with the TT technique and is feasible for IgG anti-A/B antibody titration in ABOi-LKT recipients. This article is protected by copyright. All rights reserved.

Alloyed Cu is known to inhibit the growth of pitting corrosion on stainless steels after pitting initiation. The Cu2+ dissolved from the stainless steel matrix acts as an inhibitor in acidic chloride environments which is formed in pits. The Cu2+ suppresses active dissolution rate inside the pits1. This suppression effect is supposed to be also effective for active dissolution which promotes crevice corrosion. Therefore, if it is possible to introduce Cu2+ to the inside of a crevice from the outside, introduced Cu2+ is supposed to inhibit crevice corrosion on stainless steel. However, diffusion of ions between inside and outside of the crevice is restricted by its geometry, and Cu2+ does not migrate to inside of a crevice according to the electroneutrality principle. [Cu (EDTA (ethylenediaminetetraacetic acid))] 2− is a chelated Cu2+ which has negative charge, and is expected to migrate to inside of a crevice from the outside by electrochemical migration. In addition, it is reported that Cu2+ in [Cu(EDTA)]2− could be easily substituted by Fe2+ in low pH2. Therefore, Cu2+ is considered to be introduced to the inside of the crevice as [Cu(EDTA)]2−, and to affect crevice corrosion of stainless steel. In this study, in situ observation of an inside of the crevice3 was performed to analyze the effect of [Cu(EDTA)]2− on crevice corrosion. Type 316L stainless steel was used as specimen. The crevice corrosion tests were conducted in a flow cell3. 0.1 M NaCl or 0.1 M NaCl-10 mM [Cu(EDTA)]2− was flowed through the flow cell. The crevice was made by pressing the glass plate on the specimen surface. The specimen surface was perfused with 0.1 M NaCl before the glass plate was pressed. Therefore, inside of the crevice was filled with 0.1 M NaCl. The specimen was polarized at 0.35 V (vs. Ag/AgCl, Sat. KCl). After the crevice corrosion initiation, the different propagation morphology was observed between 2 conditions. In the case of 0.1 M NaCl, the gas bubbles were observed with corrosion propagation. On the other hand, little gas bubbles were observed in the case of 0.1 M NaCl-10 mM [Cu(EDTA)]2−. The gas bubbles observed in 0.1 M NaCl were considered to be H2 generated by the H+ reduction reaction inside the crevice. It was reported that the reduction reaction inside the crevice was changed by migrated anions which reacted with H+ inside the crevice3. Therefore, the results indicate [Cu(EDTA)]2− migrated to the inside of the crevice from the outside, and affected the crevice corrosion morphology. References T. Sourisseau, E. Chauveu, and B. Baroux, Corros. Sci., 47, 1097-1117(2005). W. Guan, B. Zhang, S. Tian, and X. Zhao, Appl. Catal. B, 227, 252-257 (2018). T. Aoyama, Y. Sugawara, I. Muto, and N. Hara, J. Electrochem. Soc., 166, C250-C260 (2019).

The present study addresses the microbiome of the first whale fall (YOKO 16) that has been described in the deep sea in the southern Atlantic Ocean (Sao Paulo Plateau; 4204 m depth), in terms of its metabolic uniqueness. Sets of ten thousand protein sequences from YOKO 16 and 29 public domain metagenomes (SRA and GenBank databases) that represent various marine, terrestrial and gut-associated microbial communities were analyzed. The determination of protein functionality, based on the KAAS server, indicated that the YOKO 16 microbiome has industrially-relevant proteins, such as proteases and lipases, that have low similarity (~50%) with previously-described enzymes. The amino acid usage in the YOKO 16 protein sequences (based on blastp and Clustal analysis) revealed a pattern of preference similar to that of extremophiles, with an increased usage of polar, charged and acidic amino acids and a decreased usage of nonpolar residues. We concluded that the targeted microbiome is of potential biotechnological use, which justifies the allocation of resources for the discovery of enzymes in deep-sea whale fall communities.

Outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) for hepatitis-associated aplastic anemia have not been fully evaluated. In the present study, the outcomes of 37 adult patients with hepatitis-associated aplastic anemia who underwent allogeneic HSCT were retrospectively analyzed using the registry database of Japan Society for Hematopoietic Cell Transplantation. The median age of the patients was 24 years (range, 16-61). The median period between diagnosis of hepatitis-associated aplastic anemia and HSCT was 6.0 months (range, 0.5-430.8). Stem cell sources were bone marrow (N = 19) or peripheral blood stem cells (N = 5) from an HLA-identical sibling or bone marrow (N = 11) and cord blood (N = 2) from an unrelated donor. The majority of conditioning regimens were fludarabine-based or high-dose cyclophosphamide-based. In all but 2 cases of early death, neutrophil engraftment was achieved. At the time of analysis, 32 patients were alive, with a median follow-up of 54.1 months. Five-year overall and failure-free survival rates were 86.0% (95% CI, 69.4-93.9%) and 75.0% (95% CI, 57.4-86.2%), respectively. Despite the heterogeneity in transplant procedures in a small number of patients, these results suggest that allogeneic HSCT is safe for use in hepatitis-associated aplastic anemia with a low rate of transplant-related mortality.

We have isolated several poly 3-hydroxybutyrate-co-3-hydroxyhexanoate (PHBH) degradable bacteria from the deep-sea sediment samples. The isolated microbes were involved into α-, γ- Proteobacteria g...

The boiling nitric acid solution containing highly oxidizing cations dissolved from the spent nuclear fuels corrodes stainless steels because of the nobler corrosion potential and their fast reduct...

The utility of convolutional neural networks (CNNs) for sex estimation of the pelvis was evaluated using depth images generated from reconstructed three-dimensional (3D) computed tomography images. The 3D volume data were normalized by a homologous modeling technique to create polygon data with identical topology, then captured images for learning and testing. The neural networks were trained via transfer learning. As a result, a correct assignment rate >90% was obtained in most trials. The frontal view of the pelvis with 60-degree inclination achieved the best results. Selecting samples close to the average images of the sex was effective for training.

We characterized a protease of the M4 family from the cold-adapted Vibrio sp. Pr21 that was isolated from seawater at 320-m deep in Sagami Bay, Japan, and named it as PR protease based on the strain name Pr21. The PR protease had activities at 10–60 °C and 0.1–350 MPa, with the optimal temperature and pressure at 40 °C and 250 MPa. The mutant 10C9 (Q301P) obtained by error-prone PCR had higher activities than the wild-type enzyme at 10–60 °C, and the Q301P mutation contributed to the increase of the activity. The specific activity value of 10C9 was also higher than that of the wild-type enzyme at 0.1–200 MPa, but the specific activity ratios (1.28–1.59) of 10C9/wild-type enzyme at 50–200 MPa at 30 °C were smaller than those at 10–60 °C (1.73–4.39) at 0.1 MPa. The catalytic efficiency value of 10C9 was lower than that of the wild-type enzyme at 200 MPa. The homology models of PR protease suggested that the side chain of Q301 was hydrogen-bonded with the carbonyl oxygen atom of the main chain of N234 in the wild-type enzyme, and P301 had no contact with N234 in 10C9. The break of the hydrogen bond in 10C9 might strengthen the increase of the flexibility of the β-sheet near the substrate binding pocket under high-temperature conditions, whereas the flexibility of the β-sheet in 10C9 might be moderately increased compared to that in the wild-type enzyme under high-pressure conditions.

We previously found that the enzymatic activity of 3-isopropylmalate dehydrogenase from the obligatory piezophilic bacterium Shewanella benthica strain DB21MT-2 (SbIPMDH) was pressure-tolerant up to 100 MPa, but that from its atmospheric congener S. oneidensis strain MR-1 (SoIPMDH) was pressure-sensitive. Such characteristics were determined by only one amino acid residue at position 266, serine (SoIPMDH) or alanine (SbIPMDH) [Y. Hamajima et al. Extremophiles 20: 177, 2016]. In this study, we investigated the structural stability of these enzymes. At pH 7.6, SoIPMDH was slightly more stable against hydrostatic pressure than SbIPMDH, contrary to the physiological pressures of their normal environments. Pressure unfolding of these IPMDHs followed a two-state unfolding model between a native dimer and two unfolded monomers, and the dimer structure was pressure-tolerant up to 200 MPa, employing a midpoint pressure of 245.3 ± 0.1 MPa and a volume change of −225 ± 24 mL mol−1 for the most unstable mutant, SbIPMDH A266S. Thus, their pressure-dependent activity did not originate from structural perturbations such as unfolding or dimer dissociation. Conversely, urea-induced unfolding of these IPMDHs followed a three-state unfolding model, including a dimer intermediate. Interestingly, the first transition was strongly pH-dependent but pressure-independent; however, the second transition showed the opposite pattern. Obtained volume changes due to urea-induced unfolding were almost equal for both IPMDHs, approximately +10 and −30 mL mol−1 for intermediate formation and dimer dissociation, respectively. These results indicated that both IPMDHs have similar structural stability, and a pressure-adaptation mechanism was provided for only the enzymatic activity of SbIPMDH.

The water in Earth’s mantle is closely related with plate subduction and volcanism. Recent studies revealed that the mantle wedge corner at approximately 30 km depth holds high-pressure water, where many slow earthquakes occur. To quantify how such water behaves during slow earthquakes helps us understand the mechanisms of these earthquakes and (eventually) a part of the long-term water cycle between the interior and surface of the Earth. However, little evidence has thus far been reported on the transient flows of such deep water. Here, we report anomalous, negative mass anomalies during two recent long-term slow slip events in the Tokai area in Japan, which were detected by absolute gravity measurements over 20 years. We present a poroelastic fluid flow model assuming a localized deformation within the fault fracture zone. The model can reproduce the gravity change with a permeability range between those suggested by laboratory experiments and numerical simulations of slow earthquakes.

Identification of specific drug targets is very important for cancer therapy. We recently identified zinc finger protein of the cerebellum 5 (ZIC5) as a factor that promotes melanoma aggressiveness by platelet-derived growth factor D (PDGFD) expression. However, its roles in other cancer types remain largely unknown. Here we determined the roles of ZIC5 in prostate cancer (PCa) and colorectal cancer (CRC) cells. Results showed that ZIC5 was highly expressed in CRC and dedifferentiated PCa tissues, whereas little expression was observed in relevant normal tissues. Knockdown of ZIC5 decreased proliferation of several PCa and CRC cell lines with induction of cell death. ZIC5 knockdown significantly suppressed PDGFD expression transcriptionally, and PDGFD suppression also decreased proliferation of PCa and CRC cell lines. In addition, suppression of ZIC5 or PDGFD expression decreased levels of phosphorylated focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) which are associated with PCa and CRC aggressiveness. Furthermore, knockdown of ZIC5 or PDGFD enhanced death of PCa and CRC cells induced by the anti-cancer drugs docetaxel or oxaliplatin, respectively. These results suggest that ZIC5 and PDGFD promote survival of PCa and CRC cells by enhancing FAK and STAT3 activity, and that the roles of ZIC5 are consistent across several cancer types.

Deep-sea bacteria can produce various biotechnologically relevant enzymes due to their adaptations to high pressures and low temperatures. To identify such enzymes, we have sequenced the genome of the polycaprolactone-degrading bacterium Moritella sp. JT01, isolated from sediment samples from Japan Trench (6957 m depth), using a Illumina HiSeq2000 sequencer (12.1 million paired-end reads) and CLC Genomics Workbench (version 6.5.1) for the assembly, resulting in a 4.83-Mb genome (42 scaffolds). The genome was annotated using Rapid Annotation using Subsystem Technology (RAST), Protein Homology/analogY Recognition Engine V 2.0 (PHYRE2), and BLAST2Go, revealing 4439 protein coding sequences and 101 RNAs. Gene products with industrial relevance, such as lipases (three) and esterases (four), were identified and are related to bacterium's ability to degrade polycaprolactone. The annotation revealed proteins related to deep-sea survival, such as cold-shock proteins (six) and desaturases (three). The presence of secondary metabolite biosynthetic gene clusters suggests that this bacterium could produce nonribosomal peptides, polyunsaturated fatty acids, and bacteriocins. To demonstrate the potential of this genome, a lipase was cloned an introduced into Escherichia coli. The lipase was purified and characterized, showing activity over a wide temperature range (over 50% at 20-60 °C) and pH range (over 80% at pH 6.3 to 9). This enzyme has tolerance to the surfactant action of sodium dodecyl sulfate and shows 30% increased activity when subjected to a working pressure of 200 MPa. The genomic characterization of Moritella sp. JT01 reveals traits associated with survival in the deep-sea and their potential uses in biotechnology, as exemplified by the characterized lipase.

Fifteen actinomycetes were isolated from deep-sea sediments in Calyptogena communities in the Japan Trench (depth: 5352 m) and Sagami Bay (depth: 1171 m) under incubation at 4–37 °C. A relatively large number of colonies were obtained with incubation at 30 °C from the deep-sea sediment of Sagami Bay. We found Rhodococcus, Nocardioides and Micromonospora actinomycetes in both sediments, and additionally isolated an Aeromicrobium actinomycete from the Japan Trench and Streptomyces and Actinomadura actinomycetes from Sagami Bay. Actinomadura sp. DS-MS-114 isolated from the deep-sea sediment in Sagami Bay produced an antibacterial compound against Staphylococcus aureus NBRC12732T. Structural analysis by liquid chromatography–high-resolution electrospray ionization mass spectrometry (LC-HRESIMS) and proton nuclear magnetic resonance (1H-NMR) indicated that the antibacterial compound was 5,6-dihydro-1,8-dihydroxy-3-methylbenz[a]anthracene-7,12-quinone, which is known as an immunosuppressant. The deep-sediment in the Calyptogena community may be useful as a source of various actinomycetes producing physiologically active substances.

New bioactive substances were identified from several marine actinomycetes strains by LC-HRESI-MS based non-targeted metabolomics. A new siderophore and its derivative, named fradiamines A and B, were isolated from the extract of the deep-sea actinomycetes Streptomyces fradiae MM456M-mF7 by Diaion CHP-20P, Sephadex LH-20 column chromatography and HPLC. Fradiamine A was a new compound, but fradiamine B was previously patented as a sweetness enhancer. Their structures were determined by NMR and LC-HRESI-MS/MS analysis. Fradiamines A and B contained two alkyl amines asymmetrically bonded to citrate, a type of structure derived from actinomycetes and other bacteria and rarely observed in siderophores. Fradiamines A and B showed moderate antibiotic activity against Clostridium difficile with IC50 values of 32 and 8 μg ml-1, respectively.

In allogeneic hematopoietic stem cell transplantation (allo-HSCT), the effects of patient and donor human leukocyte antigen (HLA) matching status on graft-versus-host disease (GVHD) have been extensively elucidated, but the effects of specific HLAs on acute GVHD remain unclear. Using data from a Japanese registry, we retrospectively analyzed 4392 patients with leukemia or myelodysplastic syndrome who received transplants from HLA-identical sibling donors to investigate the effects of HLAs on acute GVHD. From unbiased searches of HLA-A, -B, and -DR, HLA-B60 was significantly associated with an increased risk of grades II-IV acute GVHD (HR, 1.34; 95% CI, 1.13-1.59; P = 0.001). In contrast, HLA-B62 was significantly associated with a decreased risk of grades II-IV (HR, 0.73; 95% CI, 0.62-0.87; P 0.001) and III-IV acute GVHD (HR, 0.63; 95% CI, 0.46-0.87; P = 0.005). The risk of leukemia relapse was significantly higher in HLA-B62-positive patients than in HLA-B62-negative patients (HR, 1.23; 95% CI, 1.05-1.43; P = 0.01). Both HLA-B60 and -B62 did not affect overall survival. The findings of this study may by implication suggest the possibility that the effects of specific HLAs on transplant outcomes may reflect inherent biological features, and thus consideration of specific HLAs may be helpful to predict transplant outcomes.

Shewanella benthica DB21MT-2 is an obligate piezophilic bacterium isolated from the sediment of the Mariana Trench at depth of 10,898 m. Here we report the complete genome sequence of S. benthica DB21MT-2, which consists of a single circular chromosome with the size of 4,353,505 bp and G + C content of 45.86%. Comparative genomic analysis reveals the strain possesses multiple pathways for energy generation as has been reported in other deep-sea bacteria. In addition, several genes predicted to be involved in flagellar synthesis or component of toxin-antitoxin system are specifically found in obligate piezophilic strains from hadal trench. The result is expected to provide novel information for a better understanding on bacterial obligate piezophilic life-style and on bacterial adaptation to hadal trench environment.