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Objective: This study aimed to determine if contrast medium volume (CMV) is a risk factor for acute kidney injury (AKI) during transcatheter aortic valve implantation (TAVI) via a transfemoral approach performed without major complications. All TAVI procedures performed at our institution between March 2014 and March 2018 were retrospectively reviewed. AKI was diagnosed using the Acute Kidney Injury Network classification based on the Valve Academic Research Consortium-2 definition. Procedures performed via a transapical approach and those in which circulatory dynamics failed intraoperatively were excluded., Results: Eighty-one (96.4%) of 100 patients scheduled for TAVI were enrolled; seven (8.6%) developed AKI and 74 (91.4%) did not. The serum creatinine (SCr) level was significantly higher (p < 0.05) and the estimated glomerular filtration rate was significantly lower in the AKI group (p < 0.05). The CMV was significantly higher in the AKI group (103 ml vs 84 ml, p < 0.05), as was the CMV × SCr/BW value (3.34 vs 1.49, p < 0.01). The area under the curve for CMV × SCr/BW was 0.9228 and the cut-off value was 2.99. The CMV, SCr, and estimated glomerular filtration rate affect the likelihood of AKI after transfemoral TAVI and a CMV × SCr/BW value > 2.99 accurately predicts AKI.

Background: Osteoporosis is a major side effect of aromatase inhibitors (AIs), which are greatly effective in the treatment of breast cancer. However, there are no satisfactory measures against osteoporosis. In this multicenter, randomized, comparative study, we evaluate the efficacy of denosumab for preventing loss of bone mineral density (BMD) induced by adjuvant therapy with AI s in breast cancer patients with normal BMD., Patients and Methods: The bone loss-suppressing effect of denosumab will be comparatively evaluated in postmenopausal patients scheduled to receive letrozole or anastrozole as a postoperative endocrine therapy for stage I-IIIA hormone-sensitive breast cancer and a control group. Patients will be administered letrozole 2.5 mg or anastrozole 1 mg once a day, and the treatment will be continued for 5 years unless recurrence, secondary cancer, or unacceptable toxicity develops. Patients in the denosumab group will receive a subcutaneous injection of 60 mg of denosumab every 6 months. The primary endpoint is the rate of change in the lumbar spine (L1-L4) BMD, as determined by dual-energy X-ray absorptiometry (DXA), 12 months after the start of the injection. The secondary endpoints were ETHICS AND DISSEMINATION:: The protocol was approved by the institutional review boards of Kyoto Prefectural University of Medicine and all the participating faculties. Written informed consent was obtained from all patients before registration, in accordance with the Declaration of Helsinki. Results of the study will be disseminated via publications in peer-reviewed journals., Trial Registration: Clinical Trials.gov Identifier: NCT03324932, Japan Registry of Clinical Trial (jRCT): CRB5180001.

Background/aim: Risk factors for chemotherapy-induced nausea and vomiting (CINV) with anthracycline-containing regimen for breast cancer patients remain unknown. The risk factors for CINV with FEC100 were investigated., Patients and Methods: Data on CINV events and patient backgrounds of 180 patients were collected from the first cycle of FEC100 treatment. In this regimen, patients were administered various antiemetics (ADs). The combinations of ADs were classified into four categories, while body mass index (BMI) was stratified into three categories. Risk factors were selected based on patient characteristics and combination of ADs. Risks for CINV were analyzed by univariate and multivariate analyses., Results: In the univariate analysis of nausea, BMI was a significant factor, while BMI and combination of ADs were significant in vomiting. In the multivariate analysis concerning nausea, BMI was a significant factor. In the analysis concerning vomiting, the combination of ADs and BMI were significant., Conclusion: BMI was the most important risk factor for nausea and vomiting, while the combination of ADs was for vomiting., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) is a powerful technique to visualize the distributions of biomolecules without any labeling. In MALDI-MSI experiments, the choice of matrix deposition method is important for acquiring favorable MSI data with high sensitivity and high reproducibility. Generally, manual or automated spray-coating and automated sublimation methods are used, but these methods have some drawbacks with respect to detection sensitivity, spatial resolution, and data reproducibility. Herein, we present an optimized matrix deposition method of sublimation coupled with recrystallization using 9-aminoacridine (9-AA) as a matrix capable of ionizing endogenous metabolites. The matrix recrystallization process after sublimation was optimized for the solvent concentration and reaction temperature for matrix-metabolite co-crystallization. This optimized method showed excellent reproducibility and spatial resolution compared to the automatic spray-coating method. Furthermore, the recrystallization step after sublimation remarkably improved the detectability of metabolites, including amino acids, nucleotide derivatives, and lipids, compared with the conventional sublimation method. To date, there have been no other reports of 9-AA-based sublimation combined with recrystallization. The present method provides an easy, sensitive, and reproducible matrix deposition method for MALDI-MSI of endogenous metabolites. Graphical Abstract.

Purpose: Both 18 F-FDG PET/CT and clonal circulating plasma cell (CPC) quantification are emerging tools for multiple myeloma (MM) prognostication that have been validated in recent studies. This study investigated the value of PET/CT coupled with CPC quantification for MM prognostication that may contribute to future risk-adapted treatment., Methods: We retrospectively analysed the prognostic relevance of a combination of pretreatment PET/CT findings and CPC levels in 163 consecutive patients with newly diagnosed, symptomatic MM receiving novel agents during induction therapies., Results: High-risk PET/CT findings and elevated CPC levels were defined by the presence of >3 focal lesions with or without extramedullary disease and CPCs ≥0.10% of the total mononuclear cells evaluated, respectively. Subsequently, patients were divided into three groups: PET-CPC stage I included patients with no high-risk PET/CT findings and low CPC levels; stage III included patients with high-risk PET/CT findings and high CPC levels; and stage II included the remaining patients. The three groups of patients differed significantly in terms of both progression-free survival (PFS) and overall survival (OS) (median PFS: not reached [NR] and 36.4 and 15.9 months, and median OS: NR, NR, and 40.4 months for stages I, II, and III, respectively; P < 0.001 for both PFS and OS). This system discriminated both PFS and OS even among younger (age < 75 years) or older (≥ 75 years) patients, patients with Revised International Staging System stage II or III, and patients with or without high-risk cytogenetic characteristics. In the multivariate analysis, the PET-CPC staging system remained prognostic for both PFS and OS., Conclusions: The PET-CPC staging system predicted survival outcomes independently of established risk factors in patients with newly diagnosed MM. Pretreatment 18 F-FDG PET/CT assessment combined with CPC quantification may improve the prognostication of MM and facilitate the development of novel risk-adapted approaches for MM.

Purpose: False-negative 18 F-FDG PET/CT, which is associated with low hexokinase-2 (HK2) expression in multiple myeloma (MM), is a new concept that is relevant for diagnosis and treatment response assessment. This study aimed to investigate the prognostic relevance of low HK2 expression-associated false-negative PET/CT in patients with MM., Methods: Ninety consecutive patients, with newly diagnosed MM, receiving novel agents during induction therapy were enrolled in this retrospective study. Patients were divided into three groups according to the combination of the positivity of PET/CT and whole-body diffusion-weighted magnetic resonance imaging (DWMRI), namely, negative DWMRI, false-negative PET/CT, and positive PET/CT., Results: False-negative PET/CT was observed in 12% patients who were older, had documented clinical history of smouldering MM, and showed lower HK2 expression levels than the positive PET/CT patients. False-negative PET/CT patients showed a clear trend of longer time to next treatment (TTNT) and progression-free survival (PFS) than the positive PET/CT patients (P = 0.035 and 0.071, respectively). Furthermore, TTNT and PFS of false-negative PET/CT patients were similar to those of patients without established high-risk PET/CT findings and significantly longer than those of high-risk PET/CT patients (P = 0.013 and 0.047, respectively)., Conclusions: This study showed, for the first time, that low HK2 expression-associated false-negative PET/CT was associated with relatively better prognosis in patients with newly diagnosed MM, suggesting that this phenomenon may not undermine the established PET/CT-based prognostication. Furthermore, this phenomenon may be useful for identifying patients at lower risk of disease progression among those with myelomatous lesions on DWMRI.

Daratumumab-based therapy has been shown to have significant clinical efficacy in phase 3 trials of patients with relapse or refractory multiple myeloma. Outside of clinical trials, however, clinical data on daratumumab remain limited. We reviewed medical records of patients who received daratumumab combination therapy at our institute (median age 74 years; median lines of prior therapy 4). The overall response rate was 69.4%, and 36.7% of patients achieved complete response (CR) or better. The proportion of patients who attained CR or better was significantly higher among patients with < 4 prior therapies than those with ≥ 4 (56.5% vs 19.2%, P = 0.009). Estimated median progression-free survival (PFS) was 12.4 months (95% confidence interval 8.6-not reached). The median PFS was significantly worse in patients who were refractory to bortezomib and lenalidomide and had received ≥ 4 lines of prior therapy. Twelve of 49 patients attained negative minimal residual disease. Common adverse events included hematological toxicities including neutropenia and lymphopenia; however, the rate of febrile neutropenia was low (3.8%). Infusion-related reactions occurred in 32.1% of patients, but were grade 1 or 2. Daratumumab combination therapies therefore appear to be effective and safe as salvage regimens in clinical practice, especially when used in the early phase.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors are drugs for diabetes and might prevent heart failure. In this study, we investigated the effects of tofogliflozin, an SGLT2 inhibitor, on cardiac hypertrophy and metabolism in hypertensive rats fed a high-fat diet. Dahl salt-sensitive (DS) rats, hypertensive model rats, and Dahl salt-resistant (DR) rats, non-hypertensive model rats, were fed a high-salt and high-fat diet containing tofogliflozin (0.005%) for 9 weeks to examine the effects of this drug on cardiac hypertrophy and metabolism. Tofogliflozin tended to suppress a rise of the systolic blood pressure, relative to the control, throughout the treatment period in both DR and DS rats, and significantly suppress a rise of the systolic blood pressure, relative to the control, at the 9th week in DS rats. Tofogliflozin reduced cardiac hypertrophy (heart weight/body weight) not only in DS rats but also in DR rats. Histological analysis showed that tofogliflozin significantly decreased cardiomyocyte hypertrophy and perivascular fibrosis in both DS and DR rats. Tofogliflozin significantly decreased the expression levels of genes related to cardiac hypertrophy (encoding for natriuretic peptides A and B and interleukin-6), and to cardiac fibrosis (encoding for transforming growth factor-β1 and collagen type IV), in DS rats. Recent studies have shown that hypertrophied and failing hearts shift to oxidizing ketone bodies as a significant fuel source. We also performed metabolome analysis for ventricular myocardial tissue. Tofogliflozin reduced 3-hydroxybutyrate, a ketone body, and significantly decreased the expression levels of β-hydroxybutyrate dehydrogenase 1 and 3-oxoacid CoA-transferase, which are related to ketone oxidization. In conclusion, tofogliflozin ameliorated cardiac hypertrophy and fibrosis along with reduction of ketone usage in myocardial tissue.

Purpose: No study has analyzed a sizeable cohort that comprised solely peripheral T-cell lymphoma (PTCL) patients for the diagnostic and prognostic performance of F-FDG PET/CT for bone marrow (BM) involvement. This study aimed to investigate the utility of PET/CT for the identification of BM involvement and to explore its prognostic relevance in patients with PTCL., Methods: Eighty-three consecutive patients with newly diagnosed PTCL were enrolled in this retrospective study. The diagnosis of BM involvement was confirmed on the basis of positive BM histology or disappearance of marrow abnormalities on follow-up PET/CT concurrently with a successful treatment response., Results: Of 28 patients with confirmed BM involvement, BM biopsy (BMB) and PET/CT detected an involvement in 17 and 25 patients. Among 66 patients with negative BM histology, 11 patients had BM involvement detected by PET/CT and furthermore showed significantly shorter progression-free and overall survival than patients without BM involvement. We recategorized the International Prognostic Index (IPI) risk groups based on the presence of BM involvement using the combined assessment of iliac crest marrow biopsy histology and PET/CT (PET/BMB-based IPI). PET/BMB-based IPI tended to perform better than conventional BMB-based IPI., Conclusions: This study included the largest population of PTCL patients for PET/CT evaluation of BM involvement so far. PET/CT exhibited a higher sensitivity for BM involvement than BMB. Furthermore, BM assessment using PET/CT identified patients at high risk of disease progression and mortality among those with negative BM histology, suggesting that PET/CT may have a potential to improve existing prognostic strategies in PTCL.

We constructed a xylose-utilizing Saccharomyces cerevisiae strain using endogenous xylose-assimilating genes (strain K7-XYL). Such self-cloning yeast is expected to make a great contribution to cost reduction of ethanol production processes. However, it is difficult to modify self-cloning yeast for optimal performance because the available gene source is limited. To improve the ethanol productivity of our self-cloning yeast, a kinetic model of ethanol production was constructed and sensitivity analysis was performed. Alcohol dehydrogenase (ADH1) was identified as a metabolic bottleneck reaction in the ethanol production pathway. An ADH1 overexpression strain (K7-XYL-ADH1) was constructed and evaluated in YP (yeast extract 10 g/L, peptone 20 g/L) medium containing 50 g/L xylose as the sole carbon source. Strain K7-XYL-ADH1 showed higher ethanol productivity (13.8 g/L) than strain K7-XYL (12.5 g/L). Then, K7-XYL-ADH1 was evaluated in YP medium containing 80 g/L glucose and 50 g/L xylose; however, the ethanol productivity did not change relative to that of K7-XYL (K7-XYL 46.3 g/L, K7-XYL-ADH1 45.9 g/L). We presumed that due to the presence of glucose, the internal redox balance of the cells had changed. On culturing in an aerated 5-L jar fermentor to change the internal redox balance of cells, strain K7-XYL-ADH1 showed higher ethanol productivity than K7-XYL (K7-XYL 45.0 g/L, K7-XYL-ADH1 49.4 g/L). Our results confirmed that ADH1 was a metabolic bottleneck in the ethanol production pathway. By eliminating the bottleneck, self-cloning yeast showed almost the same ethanol productivity as genetically modified yeast., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Neurolymphomatosis (NL) is a rare manifestation of non-Hodgkin lymphoma, in which malignant cells infiltrate the peripheral nerves. Most patients are treated with high-dose methotrexate (HD-MTX)-based systemic chemotherapy regimens similar to patients with central nervous system lymphoma. However, because NL is rare, the efficacy of HD-MTX is largely unknown. We reviewed medical records of patients diagnosed with NL over the past 10 years and identified 18 patients. The underlying hematological malignancy was diffuse large B-cell lymphoma (DLBCL) in 10 patients (55.6%), intravascular large B-cell lymphoma in six (33.3%), and other types in two patients. Ten patients were treated with HD-MTX-based systemic chemotherapy; the response rates with and without HD-MTX-based chemotherapy were 100% (n = 10) and 85.7% (n = 6), respectively (P = 0.41). The median progression-free and overall survival rates of patients with versus without HD-MTX treatment were 6.4 vs. 8.5 months (P = 0.97) and 13.5 vs. 8.5 months (P = 0.63), respectively. Despite the initial favorable responses, rapid disease recurrence was observed in most patients administered HD-MTX-based chemotherapy. Our observations suggest that HD-MTX-based chemotherapy may have insufficient efficacy against NL, and that other therapeutic approaches are required to improve the outcomes of patients with this rare disease.

According to fluorescent in situ hybridization, t(11;14) is the most common cytogenetic abnormality in amyloid light-chain (AL) amyloidosis, but its prevalence in patients with AL amyloidosis and concurrent multiple myeloma (MM) remains unknown. We aimed to examine the prevalence of t(11;14) and the differences in clinical characteristics of patients with t(11;14) who had AL amyloidosis with or without concurrent MM. We retrospectively analyzed 40 patients with AL amyloidosis between January 2008 and January 2018 at our institution. The prevalence of t(11;14) was significantly higher in patients with AL amyloidosis alone compared with those with concurrent MM (56.5% vs. 17.6%; P = 0.022). This study suggests that AL amyloidosis patients with concurrent MM have a lower prevalence of t(11;14) than those without MM and that the presence of t(11;14) may be associated with poor prognosis, irrespective of the presence or absence of MM.

We retrospectively explored the prognostic relevance of N-terminal pro-brain natriuretic peptide (NT-proBNP) and the association of NT-proBNP with cardiac and renal functions in 153 patients with newly diagnosed symptomatic multiple myeloma and no concomitant light chain amyloidosis who received novel agents. We also examined the usefulness of the new frailty system recently introduced by Mayo Clinic (combining age, performance status, and NT-proBNP). Patients with higher NT-proBNP levels (≥300 ng/L) had a significantly higher incidence of left ventricular diastolic dysfunction (LVDD) and myeloma-related renal insufficiency and significantly shorter overall survival (OS) than did those with lower NT-proBNP levels (<300 ng/L). NT-proBNP remained predictive of OS on multivariate analysis. Mayo Clinic's new frailty system showed excellent discrimination of OS. Furthermore, the Instrumental Activity of Daily Living (IADL) score (not evaluated in Mayo Clinic's study) predicted OS independently of this system, and a sharper discrimination of OS curves was obtained by the incorporation of IADL into this system. Our findings demonstrated that NT-proBNP levels were associated with both LVDD (as a host risk factor) and myeloma-related renal insufficiency (resulting from the disease aggressiveness) and provided predictive information regarding OS in patients with symptomatic myeloma. Furthermore, we, for the first time, validated Mayo Clinic's new frailty system. Our modification further improved Mayo Clinic's system by newly incorporating the IADL score., Competing Interests: CONFLICTS OF INTEREST The authors have no conflicts of interest.

Background: Aromatase inhibitors (AI) have been established as the gold-standard therapy for postmenopausal patients. Worldwide, adjuvant denosumab at a dose of 60 mg twice per year reduces the risk of clinical fractures in postmenopausal patients with breast cancer who received AI. However, the efficacy of denosumab in the treatment of AI-associated bone loss had not been prospectively evaluated in Japan. Previously, we reported the 12-month effect of denosumab in Japanese patients for the first time; the primary endpoint was the change in the percentage of bone mineral density (BMD) of the lumbar spine from baseline to 12 months., Methods: This secondary follow-up study prospectively evaluated the change in the percentage of BMD of the lumbar spine from baseline to 24 months. Postmenopausal women with early-stage, histologically confirmed, hormone receptor-positive, invasive breast cancer who were receiving or scheduled to receive AI were included. Denosumab was administered subcutaneously on day 1 of the study and then 6, 12, 18, and 24 months. The lumbar spine and bilateral femoral neck BMD was measured at baseline and 6, 12, 18, and 24 months., Results: At 18 and 24 months, the lumbar spine BMD increased by 5.9 and 7.0%, respectively. The femoral neck BMD also increased. Grade ≥ 2 hypocalcemia, osteonecrosis of the jaw, and atypical femoral fractures did not occur., Conclusions: Our prospective study showed that semiannual treatment with denosumab was associated with continuously increased BMD in Japanese women receiving adjuvant AI therapy for up to 24 months, regardless of prior AI treatment.

Assays for in vivo mutation are used to identify genotoxic hazards and phenotypes prone to genomic instability and cancer. The hypoxanthine guanine phosphoribosyl transferase (Hprt) gene and the phosphatidyl inositol glycan, class A (Pig-a) gene are endogenous X-linked genes that can be used as reporters of mutation in peripheral blood lymphocytes from most mammals. Here we describe methodology for measuring Hprt and Pig-a mutation in rat T-lymphocytes. The identification and selective expansion of mutant lymphocytes is based upon the phenotypic properties of Hprt- and Pig-a-deficient cells, that is, resistance to the purine analog, 6-thioguanine, or to the bacterial toxin, proaerolysin. Expanded mutants can be further analyzed by sequencing cDNA from the target transcripts for identification of small sequence alterations and by multiplex PCR analysis of genomic DNA for the detection of deletions.

Background: Recent clinical studies have shown that treatment with LCZ696, a complex containing the angiotensin receptor blocker valsartan and neprilysin inhibitor sacubitril, improves the prognosis of heart failure patients with a reduced ejection fraction. This study evaluated whether LCZ696 affects left ventricular hypertrophy, fibrosis, and hemodynamics in isoproterenol (ISO)-treated rats compared with valsartan alone., Methods: Male Wistar rats received subcutaneous saline (n = 10), subcutaneous ISO (2.4 mg/kg/day; n = 10), subcutaneous ISO + oral LCZ696 (60 mg/kg/day; n = 20) (ISO-LCZ), or subcutaneous ISO + oral valsartan (30 mg/kg/day; n = 20) (ISO-VAL) for 7 days., Results: LCZ696 and valsartan did not significantly reduce the increased heart weight/body weight ratio in rats treated with ISO. Echocardiography showed that the deceleration time shortened by ISO was restored by LCZ696 but not valsartan alone (p = 0.01 vs. the ISO group). Histological analysis showed that cardiac interstitial fibrosis increased by ISO was decreased significantly by LCZ696 but not valsartan alone (control: 0.10 ± 0.14%; ISO: 0.41 ± 0.32%; ISO-LCZ: 0.19 ± 0.23% [p < 0.01 vs. the ISO group]; ISO-VAL: 0.34 ± 0.23% [p = 0.34 vs. the ISO group]). Quantitative polymerase chain reaction showed that mRNA expression of Tgfb1, Col1a1, Ccl2, and Anp increased by ISO was significantly attenuated by LCZ696 but not valsartan alone (p < 0.05 vs. the ISO group)., Conclusions: LCZ696 improves cardiac fibrosis, but not hypertrophy, caused by continuous exposure to ISO in rats.

Because human epidermal growth factor-like receptor (HER) 2 is expressed on the surface of human pancreatic carcinoma cells to varying degrees, trastuzumab, an anti-HER2 monoclonal antibody (mAb), is expected to exert antibody-dependent, natural killer (NK) cell-mediated cytotoxicity (ADCC) against the cells. However, some reports found that the effect of trastuzumab against human pancreatic carcinoma cells was limited because most express only limited HER2. We examined whether anti-CD137 stimulating mAb could enhance trastuzumab-mediated ADCC against Panc-1, a human pancreatic cancer cell line with low HER2 expression, in vitro. Supplementation of anti-CD137 mAb could improve trastuzumab-mediated ADCC against Panc-1 which was insufficient without this stimulating antibody. The ADCC differed in individual cells, and this was related to the expression of CD137 on the surface of NK cells after trastuzumab stimulation in association with the Fcγ-RIIIA polymorphism. NK cells with Fcγ-RIIIA-VV/VF showed high levels of ADCC against Panc-1, but those with Fcγ-RIIIA-FF did not show optimal ADCC. In addition, trastuzumab-mediated ADCC against the human pancreatic cancer cell line Capan-1 with high HER2 expression was generally high and not affected by the Fcγ-RIIIA polymorphism. These results demonstrated that in Fcγ-RIIIA-VV/VF-carrying healthy individuals, trastuzumab plus αCD137 mAb could induce effective ADCC against HER2-low-expressing pancreatic cancer cell lines, and that such an approach may result in similar findings in patients with pancreatic cancer., Competing Interests: The authors have declared that no competing interests exist.

Background: Nodular lesions of the thyroid gland, including papillary thyroid carcinoma (PTC), may be difficult to diagnose by imaging, such as in ultrasonic echo testing, or by needle biopsy. Definitive diagnosis is made by pathological examination but takes several days. A more rapid and simple method to clarify whether thyroid nodular lesions are benign or malignant is needed. Fluorescence imaging with γ-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG) uses γ-glutamyltranspeptidase (GGT), a cell-surface enzyme, to hydrolyze the γ-glutamyl peptide and transfer the γ-glutamyl group. GGT is overexpressed in several cancers, such as breast, lung, and liver cancers. This imaging method is rapid and useful for detecting such cancers. In this study, we tried to develop a rapid fluorescence detection method for clinical samples of thyroid cancer, especially papillary carcinoma., Methods: Fluorescence imaging with gGlu-HMRG was performed to detect PTC using 23 surgically resected clinical samples. A portable imaging device conveniently captured white-light images and fluorescence images with blue excitation light. Hematoxylin-eosin (HE) staining was used to evaluate which fluorescent regions coincided with cancer, and immunohistochemical examination was used to detect GGT expression., Results: All 16 PTC samples exhibited fluorescence after topical application of gGlu-HMRG, whereas the normal sections of each sample showed no fluorescence. HE staining revealed that each fluorescent region corresponded to a region with carcinoma. The PTC samples also exhibited GGT expression, as confirmed by immunohistochemistry., Conclusions: All PTC samples were detected by fluorescence imaging with gGlu-HMRG. Thus, fluorescence imaging with gGlu-HMRG is a rapid, simple, and powerful detection tool for PTC., Competing Interests: The Toranomon Hospital ethics committee approved this study (recipient number: 936). We obtained informed consent from all patients.Written informed consent was obtained from the patients’ guardian/parent/next of kin for the publication of this report and any accompanying images.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Acquired resistance to therapeutic drugs is a serious problem for patients with cancer receiving systemic treatment. Experimentally, drug resistance is established in cell lines in vitro by repeated, continuous exposure to escalating concentrations of the drug; however, the precise mechanism underlying the acquired resistance is not always known. Here, it is demonstrated that the human colorectal cancer cell line DLD1 with acquired resistance to trifluridine (FTD), a key component of the novel, orally administered nucleoside analogue-type chemotherapeutic drug trifluridine/tipiracil, lacks functional thymidine kinase 1 (TK1) expression because of one nonsense mutation in the coding exon. Targeted disruption of the TK1 gene also conferred severe FTD resistance, indicating that the loss of TK1 protein expression is the primary cause of FTD resistance. Both FTD-resistant DLD1 cells and DLD1- TK1 -/- cells exhibited similar 5-fluorouracil (5-FU) sensitivity to that of the parental DLD1 line. The quantity of cellular pyrimidine nucleotides in these cells and the kinetics of thymidylate synthase ternary complex formation in 5-FU-treated cells is similar to DLD1 cells, indicating that 5-FU metabolism and cytotoxicity were unaffected. The current data provide molecular-based evidence that acquired resistance to FTD does not confer 5-FU resistance, implying that 5-FU-based chemotherapy would be effective even in tumors that become refractory to FTD during trifluridine/tipiracil treatment. Mol Cancer Res; 16(10); 1483-90. ©2018 AACR ., (©2018 American Association for Cancer Research.)

For strain improvement of Aspergillus oryzae, development of the transformation system is essential, wherein dominant selectable markers, including drug-resistant genes, are available. However, A. oryzae generally has a relatively high resistance to many antifungal drugs effective against yeasts and other filamentous fungi. In the course of the study, while investigating azole drug resistance in A. oryzae, we isolated a spontaneous mutant that exhibited high resistance to azole fungicides and found that pleiotropic drug resistance (PDR)-type ATP-binding cassette (ABC) transporter genes were upregulated in the mutant; their overexpression in the wild-type strain increased azole drug resistance. While deletion of the gene designated atrG resulted in increased azole susceptibility, double deletion of atrG and another gene (atrA) resulted in further azole hypersensitivity. Overall, these results indicate that the ABC transporters AtrA and AtrG are involved in azole drug resistance in A. oryzae.

Three-dimensional (3D) cell culture systems have been used to obtain multicellular spheroidal cell aggregates, or spheroids, from cancer cells. However, it is difficult to efficiently prepare large tumor-derived spheroids from cancer cells. To circumvent this problem, we here used a tool equipped with removal membrane, called Spheroid Catch, for the selection and enrichment of large-sized and/or size-matched spheroids from human squamous cell carcinoma (SAS cells) without loss of recovery. After a five-round process of selection and enrichment, we successfully isolated a subpopulation of SAS cells with augmented spheroid-forming capability, named eSAS: the efficiency of spheroid formation is 28.5% (eSAS) vs 16.8% (parental SAS). Notably, we found that some of eSAS cells survived after exposure of high doses of cisplatin in 3D culture. Moreover, orthotopic implantation by injecting eSAS cells into the tongues of nude mice showed reduced survival rate and increased tumor growth compared with those of nude mice injected with SAS cells. These results suggest that spheroids exhibiting properties of higher spheroid forming capacity can be efficiently collected by using Spheroid Catch. Indeed, genome-wide cDNA microarray and western blot analyses demonstrated higher mRNA and protein levels of hedgehog acyltransferase (HHAT), which is associated with stem maintenance in cell carcinoma by catalysing the N-palmitoylation of Hedgehog proteins, in eSAS cells than in SAS cells. We propose that Spheroid Catch could be useful for the study of spheroids, and potentially organoids, in the basic and clinical sciences, as an alternative method to other type of cell strainers., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing interests.

To improve the sensitivity of hydrogen detection using neutrons, a proton-polarization technique together with a high-pressure cooling method is necessary. The highest pressure (200 MPa) used in the experiment described here enabled relatively large protein crystals to be cooled without any cryoprotectants while retaining the protein structure, and it was confirmed that high-pressure-cooled crystals diffracted to nearly the same resolution as flash-cooled small crystals soaked with cryoprotectants. Dynamic nuclear polarization was used as a proton-polarization technique for protein crystals, and ∼300 mg polycrystalline protein doped with TEMPOL gave a maximum proton polarization of 22.3% at a temperature of 0.5 K in a 2.5 T magnetic field., (open access.)

Background/aim: Metabolomics is widely used for biomarker discovery, but conventional mass-spectrometry extraction procedures lose the spatial localization of metabolites. In this study, we directly analyzed breast carcinoma tissues embedded in frozen tissue microarrays (fTMAs) using MALDI mass-spectrometry imaging (MALDI-MSI)., Materials and Methods: A total of 119 breast tissues (84 carcinoma and 35 normal) were used. MSI data were extracted from each tissue., Results: Overall, 185 of 1,915 peaks which were commonly detected in 60% of target areas were subjected to further analysis. One hundred and fifty-two peaks of carcinoma showed significantly higher intensity than normal. Comparing metabolite profiles from carcinoma and normal tissues, energy charge (EC) and the sum of adenosine phosphate compound (AXP) indicated significantly higher intensities in cancerous tissues than normal. But comparisons of EC and AXP among lymph node metastasis, tumor size and tumor subtypes indicated no significant differences., Conclusion: Breast carcinoma tissues had higher EC and AXP values than normal. MALDI-MSI could be a tool for characterizing breast carcinoma., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Diagnostic imaging is important for predicting the pathological response to chemotherapy during neoadjuvant chemotherapy (NAC) and for considering the surgical management with appropriate resection after NAC. This study was performed to examine the accuracy of the present radiological imaging for predicting the pathological complete response (pCR)., Methods: From 188 patients in our previous JONIE1 Study, a randomized controlled trial comparing chemotherapy with and without zoledronic acid for patients with human epidermal growth factor receptor 2-negative breast cancer, we evaluated 122 patients whose tumor size was examined by magnetic resonance imaging or ultrasound at three points: before NAC; after administering fluorouracil, epirubicin, and cyclophosphamide; and after NAC. The maximum tumor diameter was evaluated by magnetic resonance imaging or ultrasound. Tumor reduction ratios were calculated at the same three points. The association between the radiological clinical response and the pCR was examined., Results: Among the 122 patients evaluated, there were 98 and 24 patients with luminal (Lum) and triple-negative (TN) subtypes, respectively. There were no patients who showed tumor progression after treatment. The radiological size of the tumors was finally reduced by an average of 58.4%. Clinical complete response and pCR were achieved in 22 (18.0%) and 15 (12.3%) patients, respectively. In the overall population (n = 122), the accuracy, sensitivity, and specificity for predicting pCR were 86.1%, 88.8%, and 66.7%, respectively. The negative predictive value and false-negative rate were 45.5% and 11.2%, respectively. According to subtypes, the accuracies were 83.7% and 95.8% in Lum and TN, respectively. Negative predictive value and false-negative rate were markedly different between the Lum (29.4% and 13.5%) and TN subtypes (100% and 0%), respectively., Conclusions: This randomized clinical trial demonstrated that NAC was safe for operable breast cancer patients with appropriate radiological monitoring. Radiological evaluation after NAC may be a reliable method for predicting pathological response in the TN subtype, but not in the Lum subtype., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Growth plates at each end of vertebral bodies play a pivotal role in longitudinal spinal growth. Epiphyseal closures are formed in adult humans. Although monkeys are frequently employed in bone and disc research, the age of epiphyseal closure has not been well documented. In this study, histological analyses of lumbar vertebral end plates and the surrounding tissue were performed in 11 normal cynomolgus monkeys aged approximately 9 to 15 years, and unclosed growth plate cartilage was detected in all the end plates. The data from this study constitute the first documentation of persistent vertebral growth plate cartilage in cynomolgus monkeys. The persistence of growth plate cartilage in cynomolgus monkeys approximately 15 years of age or younger, which differs from the complete epiphyseal closure exhibited in adult humans, may affect the biomechanical behavior of the spine. This is an important factor to consider in extrapolating the results of spine and intervertebral disc research using cynomolgus monkeys to adult humans.

Clinical application of the major anticancer drug, cisplatin, is limited by severe side effects, especially acute kidney injury (AKI) caused by nephrotoxicity. The detailed metabolic mechanism is still largely unknown. Here, we used an integrated technique combining mass spectrometry imaging (MSI) and liquid chromatography-mass spectrometry (LC-MS) to visualize the diverse spatiotemporal metabolic dynamics in the mouse kidney after cisplatin dosing. Biological responses to cisplatin was more sensitively detected within 24 h as a metabolic alteration, which is much earlier than possible with the conventional clinical chemistry method of blood urea nitrogen (BUN) measurement. Region-specific changes (e.g., medulla and cortex) in metabolites related to DNA damage and energy generation were observed over the 72-h exposure period. Therefore, this metabolomics approach may become a novel strategy for elucidating early renal responses to cisplatin, prior to the detection of kidney damage evaluated by conventional method., (Copyright © 2018. Published by Elsevier Inc.)

Elevated circulating uremic toxins are associated with a variety of symptoms and organ dysfunction observed in patients with chronic kidney disease (CKD). Indoxyl sulfate (IS) and p -cresyl sulfate (PCS) are representative uremic toxins that exert various harmful effects. We recently showed that IS induces metabolic alteration in skeletal muscle and causes sarcopenia in mice. However, whether organ-specific accumulation of IS and PCS is associated with tissue dysfunction is still unclear. We investigated the accumulation of IS and PCS using liquid chromatography/tandem mass spectrometry in various tissues from mice with adenine-induced CKD. IS and PCS accumulated in all 15 organs analyzed, including kidney, skeletal muscle, and brain. We also visualized the tissue accumulation of IS and PCS with immunohistochemistry and mass spectrometry imaging techniques. The oral adsorbent AST-120 prevented some tissue accumulation of IS and PCS. In skeletal muscle, reduced accumulation following AST-120 treatment resulted in the amelioration of renal failure-associated muscle atrophy. We conclude that uremic toxins can accumulate in various organs and that AST-120 may be useful in treating or preventing organ dysfunction in CKD, possibly by reducing tissue accumulation of uremic toxins., Competing Interests: The authors declare no competing financial interests.

Background: A randomized phase 2 trial in women with HER2-negative breast cancer has shown that adding zoledronic acid (ZOL) to neoadjuvant chemotherapy (CT) has potential anticancer benefits in postmenopausal and triple-negative (TN) breast cancer patients. We report the data for the secondary end point of disease-free survival (DFS)., Methods: Patients were randomly assigned to receive CT or CT + ZOL (CT-Z). All patients received four cycles of FEC100 followed by 12 cycles of paclitaxel weekly. ZOL (4 mg) was administered 3-4 times weekly for 7 wk to the CT-Z group patients. The primary end point was pathologic complete response (pCR). The secondary end points were the clinical response rates, rate of breast-conserving surgery, safety, and DFS., Results: Of the 188 patients enrolled, 95 were assigned to the CT group and 93 to the CT-Z group. DFS and overall survival were analyzed in 92 and 88 patients with the mean times of 5.15 y and 5.38 y, respectively. The 3-y DFS rate was 84.6% in the CT group and 90.8% in the CT-Z group (P = 0.188). The particular benefit from ZOL for the neoadjuvant CT seen as improvement of the pCR rate was indicated in the 3-y DFS period for TN cancer cases (CT versus CT-Z: 70.6% versus 94.1%) but not for postmenopausal cases., Conclusions: ZOL did not improve DFS when combined with CT. However, the improvement of the pCR rate translated to survival outcomes in TN breast cancer. The short-term application of ZOL may not be sufficient to improve the outcome in postmenopausal patients., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Pharmacological treatment of hypercalcemia is essential for patients with parathyroid carcinoma and intractable primary hyperparathyroidism (PHPT). Use of the calcimimetic cinacalcet hydrochloride (cinacalcet) is an option to treat such patients. We investigated the efficacy and safety of cinacalcet in Japanese patients with parathyroid carcinoma and intractable PHPT. Five Japanese patients with parathyroid carcinoma and two with intractable PHPT were enrolled in an open-label, single-arm study consisting of titration and maintenance phases. Cinacalcet doses were titrated until the albumin-corrected serum calcium concentration decreased to 10.0 mg/dL or less or until dose escalation was considered not necessary or feasible. Serum calcium concentration at the baseline was 12.1 ± 1.3 mg/dL (mean ± standard deviation; range 10.4-14.6 mg/dL) and decreased to 10.1 ± 1.6 mg/dL (range 8.6-13.3 mg/dL) at the end of the titration phase with cinacalcet at a dosage of up to 75 mg three times a day. At the end of the titration phase, at least a 1 mg/dL reduction in serum calcium concentration from the baseline was observed in five patients (three with carcinoma and two with PHPT), and it decreased to the normocalcemic range in five patients (three with carcinoma and two with PHPT). Common adverse events were nausea and vomiting. One patient discontinued participation in the study because of an adverse event, liver disorder. Cinacalcet effectively relieved hypercalcemia in 60% of the Japanese patients with parathyroid carcinoma and might be effective in those with intractable PHPT. The drug might be tolerable and safe at a dosage of at most 75 mg three times a day.

Low-molecular-weight phytochemicals have health benefits and reduce the risk of diseases, but the mechanisms underlying their activities have remained elusive because of the lack of a methodology that can easily visualize the exact behavior of such small molecules. Recently, we developed an in situ label-free imaging technique, called mass spectrometry imaging, for visualizing spatially-resolved biotransformations based on simultaneous mapping of the major bioactive green tea polyphenol and its phase II metabolites. In addition, we established a mass spectrometry-based metabolic profiling technique capable of evaluating the bioactivities of diverse green tea extracts, which contain multiple phytochemicals, by focusing on their compositional balances. This methodology allowed us to simultaneously evaluate the relative contributions of the multiple compounds present in a multicomponent system to its bioactivity. This review highlights small molecule-sensing techniques for visualizing the complex behaviors of herbal components and linking such information to an enhanced understanding of the functionalities of multicomponent medicinal herbs., Competing Interests: The authors declare no conflict of interest.

Vascular medial calcification is often observed in patients with arteriosclerosis. It is also associated with systolic hypertension, wide pulse pressure, and fluctuation of blood pressure, which results in cardiovascular events. Eicosapentaenoic acid (EPA) has been shown to suppress vascular calcification in previous animal experiments. We investigated the inhibitory effects of EPA on Wnt signaling, which is one of the important signaling pathways involved in vascular calcification. Intake of food containing 5% EPA resulted in upregulation of the mRNA expression of Klotho , an intrinsic inhibitor of Wnt signaling, in the kidneys of wild-type mice. Expression levels of β-catenin, an intracellular signal transducer in the Wnt signaling pathway, were increased in the aortas of Klotho mutant ( kl / kl ) mice compared to the levels in the aortas of wild-type mice. Wnt3a or BIO, a GSK-3 inhibitor that activates β-catenin signaling, upregulated mRNA levels of AXIN2 and LEF1 , Wnt signaling marker genes, and RUNX2 and BMP4 , early osteogenic genes, in human aorta smooth muscle cells. EPA suppressed the upregulation of AXIN2 and BMP4 . The effect of EPA was cancelled by T0070907, a PPARγ inhibitor. The results suggested that EPA could suppress vascular calcification via the inhibition of Wnt signaling in osteogenic vascular smooth muscle cells via PPARγ activation., Competing Interests: The authors have no conflict of interest to declare.

Background: The klotho gene was identified as an "aging-suppressor" gene that accelerates arterial calcification when disrupted. Serum and vascular klotho levels are reduced in patients with chronic kidney disease, and the reduced levels are associated with arterial calcification. Intake of eicosapentaenoic acid (EPA), an n-3 fatty acid, reduces the risk of fatal coronary artery disease. However, the effects of EPA on arterial calcification have not been fully elucidated. The aim of this study was to determine the effect of EPA on arterial calcification in klotho mutant mice., Methods and Results: Four-week-old klotho mutant mice and wild-type (WT) mice were given a diet containing 5% EPA (EPA food, klotho and WT: n = 12, each) or not containing EPA (control food, klotho and WT: n = 12, each) for 4 weeks. Calcium volume scores of thoracic and abdominal aortas assessed by computed tomography were significantly elevated in klotho mice after 4 weeks of control food, but they were not elevated in klotho mice after EPA food or in WT mice. Serum levels of EPA and resolvin E1, an active metabolite of EPA, in EPA food-fed mice were significantly increased compared to those in control food-fed mice. An oxidative stress PCR array followed by quantitative PCR revealed that NADPH oxidase-4 (NOX4), an enzyme that generates superoxide, gene expression was up-regulated in arterial smooth muscle cells (SMCs) of klotho mice. Activity of NOX was also significantly higher in SMCs of klotho mice than in those of WT mice. EPA decreased expression levels of the NOX4 gene and NOX activity. GPR120, a receptor of n-3 fatty acids, gene knockdown by siRNA canceled effects of EPA on NOX4 gene expression and NOX activity in arterial SMCs of klotho mice., Conclusions: EPA prevents arterial calcification together with reduction of NOX gene expression and activity via GPR120 in klotho mutant mice.

Three types of bending tests -the 3-point bending test, 4-point bending test and biaxial flexural test- were performed to examine the correlations among the testing methods for dental hard resins (HRs). The results for 5 HRs showed that the bending strengths in descending order were: biaxial flexural strength>3-point bending strength>4-point bending strength. Regression analysis of the test methods indicated that the coefficients of determination were large for all test methods; the largest was for the combination of the 4-point bending test and biaxial flexural tests. The Weibull moduli ranged from 5.42 to 10.61, and a similar descending-order trend was found in the Weibull characteristic strength (S 0 ) of the test methods. The biaxial flexural test method is thus a valid test of the flexural strength of dental hard resins.

Mapping brain function to brain structure is a fundamental task for neuroscience. For such an endeavour, the Drosophila larva is simple enough to be tractable, yet complex enough to be interesting. It features about 10,000 neurons and is capable of various taxes, kineses and Pavlovian conditioning. All its neurons are currently being mapped into a light-microscopical atlas, and Gal4 strains are being generated to experimentally access neurons one at a time. In addition, an electron microscopic reconstruction of its nervous system seems within reach. Notably, this electron microscope-based connectome is being drafted for a stage 1 larva - because stage 1 larvae are much smaller than stage 3 larvae. However, most behaviour analyses have been performed for stage 3 larvae because their larger size makes them easier to handle and observe. It is therefore warranted to either redo the electron microscopic reconstruction for a stage 3 larva or to survey the behavioural faculties of stage 1 larvae. We provide the latter. In a community-based approach we called the Ol 1 mpiad, we probed stage 1 Drosophila larvae for free locomotion, feeding, responsiveness to substrate vibration, gentle and nociceptive touch, burrowing, olfactory preference and thermotaxis, light avoidance, gustatory choice of various tastants plus odour-taste associative learning, as well as light/dark-electric shock associative learning. Quantitatively, stage 1 larvae show lower scores in most tasks, arguably because of their smaller size and lower speed. Qualitatively, however, stage 1 larvae perform strikingly similar to stage 3 larvae in almost all cases. These results bolster confidence in mapping brain structure and behaviour across developmental stages., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Although understanding their chemical composition is vital for accurately predicting the bioactivity of multicomponent drugs, nutraceuticals, and foods, no analytical approach exists to easily predict the bioactivity of multicomponent systems from complex behaviors of multiple coexisting factors. We herein represent a metabolic profiling (MP) strategy for evaluating bioactivity in systems containing various small molecules. Composition profiles of diverse bioactive herbal samples from 21 green tea extract (GTE) panels were obtained by a high-throughput, non-targeted analytical procedure. This employed the matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) technique, using 1,5-diaminonaphthalene (1,5-DAN) as the optical matrix for detecting GTE-derived components. Multivariate statistical analyses revealed differences among the GTEs in their antioxidant activity, oxygen radical absorbance capacity (ORAC). A reliable bioactivity-prediction model was constructed to predict the ORAC of diverse GTEs from their compositional balance. This chemometric procedure allowed the evaluation of GTE bioactivity by multicomponent rather than single-component information. The bioactivity could be easily evaluated by calculating the summed abundance of a few selected components that contributed most to constructing the prediction model. 1,5-DAN-MALDI-MS-MP, using diverse bioactive sample panels, represents a promising strategy for screening bioactivity-predictive multicomponent factors and selecting effective bioactivity-predictive chemical combinations for crude multicomponent systems.

Ad4-binding protein/steroidogenic factor 1 (Ad4BP/SF-1), a member of the nuclear receptor superfamily, is expressed in steroidogenic cells and regulates all steroidogenic gene expression. We recently employed mRNA and chromatin immunoprecipitation sequence (ChIP-seq) to demonstrate that Ad4BP/SF-1 directly regulates the expression of nearly all glycolytic genes. The pentose phosphate pathway (PPP) contributes to the production of nicotinamide adenine dinucleotide phosphate (NADPH). Although the expression of PPP genes and intracellular NADPH were decreased by Ad4BP/SF-1 knockdown, these genes were not the direct targets of Ad4BP/SF-1. This study therefore investigates whether Ad4BP/SF-1 directly regulates genes implicated in NADPH production. Examination of previously published data sets of mRNA sequence (mRNA-seq) and ChIP-seq strongly suggested a possibility that other NADPH-producing genes, such as malic enzyme 1 (Me1) and methylenetetrahydrofolate dehydrogenase 2 (Mthfd2), are the direct targets of Ad4BP/SF-1. Reporter gene assays and determination of intracellular NADPH concentration supported the notion that Ad4BP/SF-1 regulates NADPH production by regulating these genes. NADPH is required for macromolecule synthesis of compounds such as steroids, and for detoxification of reactive oxygen species. When synthesizing steroid hormones, steroidogenic cells consume NADPH through enzymatic reactions mediated by steroidogenic P450s. NADPH is also consumed through elimination of reactive oxygen species produced as the byproducts of the P450 reactions. Overall, Ad4BP/SF-1 potentially maintains the intracellular NADPH level through cooperative regulation of genes involved in the biological processes for consumption and supply.

Relative to other nutrients, less is known about how animals sense amino acids and how behaviour is organized accordingly. This is a significant gap in our knowledge because amino acids are required for protein synthesis - and hence for life as we know it. Choosing Drosophila larvae as a case study, we provide the first systematic analysis of both the preference behaviour for, and the learning of, all 20 canonical amino acids in Drosophila We report that preference for individual amino acids differs according to the kind of amino acid, both in first-instar and in third-instar larvae. Our data suggest that this preference profile changes across larval instars, and that starvation during the third instar also alters this profile. Only aspartic acid turns out to be robustly attractive across all our experiments. The essentiality of amino acids does not appear to be a determinant of preference. Interestingly, although amino acids thus differ in their innate attractiveness, we find that all amino acids are equally rewarding. Similar discrepancies between innate attractiveness and reinforcing effect have previously been reported for other tastants, including sugars, bitter substances and salt. The present analyses will facilitate the ongoing search for the receptors, sensory neurons, and internal, homeostatic amino acid sensors in Drosophila ., (© 2017. Published by The Company of Biologists Ltd.)

Pancreatic cancer progression involves components of the tumor microenvironment, including stellate cells, immune cells, endothelial cells, and the extracellular matrix. Although peripancreatic fat is the main stromal component involved in extra-pancreatic invasion, its roles in local invasion and metastasis of pancreatic cancer remain unclear. This study investigated the role of adipose tissue in pancreatic cancer progression using genetically engineered mice (Pdx1-Cre; LSL-KrasG12D; Trp53R172H/+) and an in vitro model of organotypic fat invasion. Mice fed a high fat diet had significantly larger primary pancreatic tumors and a significantly higher rate of distant organ metastasis than mice fed a standard diet. In the organotypic fat invasion model, pancreatic cancer cell clusters were smaller and more elongated in shape and showed increased fibrosis. Adipose tissue-derived conditioned medium enhanced pancreatic cancer cell invasiveness and gemcitabine resistance, as well as inducing morphologic changes in cancer cells and increasing the numbers of lipid droplets in their cytoplasm. The concentrations of oleic, palmitoleic, and linoleic acids were higher in adipose tissue-derived conditioned medium than in normal medium, with these fatty acids significantly enhancing the migration of cancer cells. Mature adipocytes were smaller and the concentration of fatty acids in the medium higher when these cells were co-cultured with cancer cells. These findings indicate that lipolytic and fibrotic changes in peripancreatic adipose tissue enhance local invasiveness and metastasis via adipocyte-released fatty acids. Inhibition of fatty acid uptake by cancer cells may be a novel therapy targeting interactions between cancer and stromal cells.

Information on spatiotemporal metabolic behavior is indispensable for a precise understanding of physiological changes and responses, including those of ripening processes and wounding stress, in fruit, but such information is still limited. Here, we visualized the spatial distribution of metabolites within tissue sections of tomato (Solanum lycopersicum L.) fruit using a matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) technique combined with a matrix sublimation/recrystallization method. This technique elucidated the unique distribution patterns of more than 30 metabolite-derived ions, including primary and secondary metabolites, simultaneously. To investigate spatiotemporal metabolic alterations during physiological changes at the whole-tissue level, MALDI-MSI was performed using the different ripening phenotypes of mature green and mature red tomato fruits. Although apparent alterations in the localization and intensity of many detected metabolites were not observed between the two tomatoes, the amounts of glutamate and adenosine monophosphate, umami compounds, increased in both mesocarp and locule regions during the ripening process. In contrast, malate, a sour compound, decreased in both regions. MALDI-MSI was also applied to evaluate more local metabolic responses to wounding stress. Accumulations of a glycoalkaloid, tomatine, and a low level of its glycosylated metabolite, esculeoside A, were found in the wound region where cell death had been induced. Their inverse levels were observed in non-wounded regions. Furthermore, the amounts of both compounds differed in the developmental stages. Thus, our MALDI-MSI technique increased the understanding of the physiological changes and responses of tomato fruit through the determination of spatiotemporally resolved metabolic alterations. Graphical abstract ᅟ., Competing Interests: The authors declare that they have no conflict of interest.

Successful treatment of aspergillosis caused by Aspergillus fumigatus is threatened by an increasing incidence of drug resistance. This situation is further complicated by the finding that strains resistant to azoles, the major antifungal drugs for aspergillosis, have been widely disseminated across the globe. To elucidate mechanisms underlying azole resistance, we identified a novel transcription factor that is required for normal azole resistance in Aspergillus fungi including A. fumigatus, Aspergillus oryzae, and Aspergillus nidulans. This fungal-specific Zn2-Cys6 type transcription factor AtrR was found to regulate expression of the genes related to ergosterol biosynthesis, including cyp51A that encodes a target protein of azoles. The atrR deletion mutant showed impaired growth under hypoxic conditions and attenuation of virulence in murine infection model for aspergillosis. These results were similar to the phenotypes for a mutant strain lacking SrbA that is also a direct regulator for the cyp51A gene. Notably, AtrR was responsible for the expression of cdr1B that encodes an ABC transporter related to azole resistance, whereas SrbA was not involved in the regulation. Chromatin immunoprecipitation assays indicated that AtrR directly bound both the cyp51A and cdr1B promoters. In the clinically isolated itraconazole resistant strain that harbors a mutant Cyp51A (G54E), deletion of the atrR gene resulted in a hypersensitivity to the azole drugs. Together, our results revealed that AtrR plays a pivotal role in a novel azole resistance mechanism by co-regulating the drug target (Cyp51A) and putative drug efflux pump (Cdr1B)., Competing Interests: The authors have declared that no competing interests exist.

Detection of in vivo mutation is important for evaluating the health risks associated with chemicals. The Pig-a in vivo gene mutation assay has been developed over the last decade for this purpose. Most approaches for the assay, however, measure cells with a Pig-a mutant phenotype in erythrocytes from the peripheral blood, with the mutations causing the phenotype being difficult to determine directly. This chapter describes a procedure for detecting mutations in the Pig-a gene of phenotypically mutant mouse bone marrow erythroids, the precursors of peripheral blood erythrocytes. The strategy for molecular analysis of Pig-a gene mutation includes enrichment of GPI-anchor deficient cells with a cell sorter followed by a cloning and sequencing of Pig-a cDNAs.

Cyclin G1 (CycG1) and Cyclin G2 (CycG2) play similar roles during the DNA damage response (DDR), but their detailed roles remain elusive. To investigate their distinct roles, we generated knockout mice deficient in CycG1 (G1KO) or CycG2 (G2KO), as well as double knockout mice (DKO) deficient in both proteins. All knockouts developed normally and were fertile. Generation of mouse embryonic fibroblasts (MEFs) from these mice revealed that G2KO MEFs, but not G1KO or DKO MEFs, were resistant to DNA damage insults caused by camptothecin and ionizing radiation (IR) and underwent cell cycle arrest. CycG2, but not CycG1, co-localized with γH2AX foci in the nucleus after γ-IR, and γH2AX-mediated DNA repair and dephosphorylation of CHK2 were delayed in G2KO MEFs. H2AX associated with CycG1, CycG2, and protein phosphatase 2A (PP2A), suggesting that γH2AX affects the function of PP2A via direct interaction with its B'γ subunit. Furthermore, expression of CycG2, but not CycG1, was abnormal in various cancer cell lines. Kaplan-Meier curves based on TCGA data disclosed that head and neck cancer patients with reduced CycG2 expression have poorer clinical prognoses. Taken together, our data suggest that reduced CycG2 expression could be useful as a novel prognostic marker of cancer.

Evaluating the severity of depression (SOD), especially suicidal ideation (SI), is crucial in the treatment of not only patients with mood disorders but also psychiatric patients in general. SOD has been assessed on interviews such as the Hamilton Rating Scale for Depression (HAMD)-17, and/or self-administered questionnaires such as the Patient Health Questionnaire (PHQ)-9. However, these evaluation systems have relied on a person's subjective information, which sometimes lead to difficulties in clinical settings. To resolve this limitation, a more objective SOD evaluation system is needed. Herein, we collected clinical data including HAMD-17/PHQ-9 and blood plasma of psychiatric patients from three independent clinical centers. We performed metabolome analysis of blood plasma using liquid chromatography mass spectrometry (LC-MS), and 123 metabolites were detected. Interestingly, five plasma metabolites (3-hydroxybutyrate (3HB), betaine, citrate, creatinine, and gamma-aminobutyric acid (GABA)) are commonly associated with SOD in all three independent cohort sets regardless of the presence or absence of medication and diagnostic difference. In addition, we have shown several metabolites are independently associated with sub-symptoms of depression including SI. We successfully created a classification model to discriminate depressive patients with or without SI by machine learning technique. Finally, we produced a pilot algorithm to predict a grade of SI with citrate and kynurenine. The above metabolites may have strongly been associated with the underlying novel biological pathophysiology of SOD. We should explore the biological impact of these metabolites on depressive symptoms by utilizing a cross species study model with human and rodents. The present multicenter pilot study offers a potential utility for measuring blood metabolites as a novel objective tool for not only assessing SOD but also evaluating therapeutic efficacy in clinical practice. In addition, modification of these metabolites by diet and/or medications may be a novel therapeutic target for depression. To clarify these aspects, clinical trials measuring metabolites before/after interventions should be conducted. Larger cohort studies including non-clinical subjects are also warranted to clarify our pilot findings., Competing Interests: The authors have declared that no competing interests exist.

Mass spectrometry imaging (MSI) visualizes the simultaneous lateral distribution of multiple compounds on sample surface. However, it is still difficult to visualize biological indices such as energy charge index from multiple compounds because of the lack of publicly available tools. Here we present MSIdV, a visualization tool for biological indices calculated from mass spectrometry imaging data, which can effectively scan a series of mass spectra and process, calculate and visualize user-defined index measures accurately with a number of signal processing features., Availability and Implementation: MSIdV is implemented in Python 2.7 and is freely available on the web at https://sourceforge.net/projects/msidv/ CONTACT: eisuke.hayakawa@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

As part of a collaborative study in the Mammalian Mutagenicity Study group of the Japanese Environmental Mutagen Society, we evaluated the in vivo mutagenicity of isopropyl p-toluenesulfonate (IPTS) using a peripheral blood Pig-a assay in rats. Pig-a mutant frequency (MF) data was obtained for both red blood cells (RBCs) and reticulocytes (RETs) at 1, 2 and 4 weeks after a single oral administration of IPTS at doses of 125, 250, or 500mg/kg. The results of the RBC Pig-a assay demonstrated that both the 250 and 500mg/kg treatment groups showed significant increases in Pig-a MF only at 4 weeks after IPTS treatment. In comparison, the PIGRET assay showed a clear and dose-related increase in Pig-a MF at 1 week after treatment, with a continuous increase until 4 weeks after treatment observed in the highest dose group. These results indicate that the both the RBC Pig-a assay and PIGRET assay can detect in vivo IPTS mutagenicity under a single dosing protocol. In particular, the PIGRET assay, which uses magnetic enrichment to analyze greater numbers of RETs in a high-throughput manner, showed an increase in Pig-a MF earlier than the RBC Pig-a assay. The PIGRET assay is also considered to be more sensitive than the RBC Pig-a assay because it exhibits a low spontaneous Pig-a MF. For this reason, the PIGRET assay clearly identified small increases in Pig-a MF as significant at the lower doses than in the RBC Pig-a assay under the conditions in this study., (Copyright © 2016. Published by Elsevier B.V.)