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The present study aimed to establish a novel isolation strategy for circulating tumor cells (CTCs) using a microcavity array (MCA) system and to evaluate the clinical significance of CTCs in hepatocellular carcinoma (HCC). We examined recovery rates of HCC cell lines spiked into whole blood in MCA assay. Circulating tumor cells were isolated from peripheral blood samples (3 mL) of 7 healthy donors (HD), 14 patients with liver cirrhosis (LC), and 31 patients with HCC using the MCA system. Additionally, we investigated the mRNA expression of liver‐specific genes in isolated CTCs using qPCR. The recovery rates were 65.1% (HepG2), 76.7% (HuH7), and 99.0% (PLC/PRF/5). In HD and patients with LC and HCC, the CTC positivity rate (CTCs ≥10) and average CTC number were as follows: HD 0% and 0.1, LC 14.3% and 5.3, HCC 54.8% and 47.6, respectively. The CTC positivity rate in HCC was significantly higher than that in LC (p, This study describes the detection of circulating tumor cells (CTCs) in the peripheral blood of hepatocellular carcinoma (HCC) patients using a newly developed microcavity array. Our results show that a large number of CTCs was detected in metastatic HCC patients, and mRNA expression of AFP, glypican‐3, EpCAM and albumin was detected in detected CTCs. Identification of positive CTCs or ALB mRNA expression can help predict poor prognosis of HCC patients.

Noninvasive diagnostics using circulating tumor cells (CTCs) are expected to be useful for decision making in precision cancer therapy. AXL, a receptor tyrosine kinase is associated with tumor progression, epithelial‐to‐mesenchymal transition (EMT), and drug resistance, and is a potential therapeutic target. However, the epithelial markers generally used for CTC detection may be not enough to detect AXL‐expressing CTCs due to EMT. Here, we evaluated the detection of AXL‐expressing CTCs using the mesenchymal marker vimentin with a microcavity array system. To evaluate the recovery of cancer cells, spike‐in experiments were performed using cell lines with varying cytokeratin (CK) or vimentin (VM) expression levels. With high CK and low VM‐expressing cell lines, PC‐9 and HCC827, the recovery rate of AXL‐expressing cancer cells was 1%‐17% using either CK or VM as markers. Whereas, with low CK and high VM‐expressing cell lines, MDA‐MB231 and H1299, it was 52%‐75% using CK and 72%‐88% using VM as a marker. For clinical evaluation, peripheral blood was collected from 20 non–small cell lung cancer patients and CTCs were detected using CK or VM as markers in parallel. Significantly more AXL‐expressing single CTCs were detected in VM‐positive than CK‐positive CTCs (P, We established the detection of AXL‐expressing in circulating tumor cells of lung cancer patients incorporating vimentin as a CTC marker in addition to cytokeratin. Significantly more AXL‐expressing CTCs were detected in patients who had prior treatment history than those did not, suggesting the involvement of AXL in drug resistance.

We report a bioinspired synthesis of 2,5-dihydropentalene-based chromophores from an aliphatic oligoketone bearing 1,3- and 1,4-diketone subunits. Unlike the natural polyketone sequence, fused five-membered rings were formed via an intramolecular aldol condensation. A subsequent Knoevenagel condensation reaction with malononitrile furnished a multiply cross-conjugated π-system with low-lying LUMO levels. Furthermore, pentalenes obtained from a non-conjugated aliphatic chain exhibited visible absorption and solid-state fluorescence.

Background Blockade of the programmed death receptor-1 (PD-1) pathway is effective against solid tumors including lung cancer. PD-ligand 1 (PD-L1) expression on tumor tissue serves as a predictive biomarker for the efficacy of PD-1 pathway blockade. Here, we evaluated the expression of PD-L1 on circulating tumor cells (CTCs) in patients with lung cancer. Materials and Methods Peripheral whole blood (3 mL) was collected from patients, and CTCs and PD-L1 expression were detected using a microcavity array (MCA) system. Immunohistochemistry for PD-L1 detection was also performed using matched tumor tissues. Results Sixty-seven patients with lung cancer were enrolled in the study between July 2015 and April 2016 at Wakayama Medical University Hospital. The characteristics of the patients were as follows: median age, 71 years (range, 39-86 years); male, 72%; stage II to III/IV, 14%/85%; non–small-cell lung cancer/small-cell lung cancer/other, 73%/21%/6%. CTCs were detected in 66 of 67 patients (median, 19; range, 0-115), and more than 5 CTCs were detected in 78% of patients. PD-L1-expressing CTCs were detected in 73% of patients, and the proportion score of PD-L1-expressing CTCs ranged from 3% to 100%, suggesting intra-patient heterogeneity of PD-L1 expression on CTCs. Tumor tissues were available from 27 patients and were immunostained for PD-L1, and no correlation was observed between tumor tissues and CTCs based on the proportion score (R2 = 0.0103). Conclusion PD-L1 expression was detectable on CTCs in patients with lung cancer, and intra-patient heterogeneity was observed. No correlation was observed between PD-L1 expression in tumor tissues and CTCs.

Purpose: We have previously reported that PD-L1-positive rate on CTCs at baseline was correlated with response to nivolumab in NSCLC patients. Here, we conducted a longitudinal follow-up investigation of PD-L1 expression on CTCs and its association with clinical outcome. Methods: For CTC detection, 3ml of peripheral blood was collected from advanced NSCLC patients treated with nivolumab at baseline and week 4, 8, 12 and 24 or until progressive disease (PD). CTCs were enriched by microcavity array system based on the difference in size and defined as DAPI-positive, CK-positive, and CD45-negative cells and evaluated for PD-L1 expression by additional immunostaining. Results: Forty-five patients were enrolled between January 2016 and January 2018 at Wakayama Medical University. The patient characteristics were as follows: median age 68 years (range, 49-86); male 75%; stage III/IV, 25/75%; adenocarcinoma/ squamous cell carcinoma/ other, 68/25/7%; partial response/stable disease/PD/not evaluable, 20/25/45/9%. At baseline, CTCs were evaluated in 44 patients (median, 13; range, 1-104) and PD-L1 positive CTCs were detected in 82% patients (median 29%; range, 0-100%), at week 4, evaluated in 31 patients (median, 4; range, 0-115) and detected in 58% patients (median, 13%; range, 0-100%), at week 8, evaluated in 16 patients (median, 11; range, 1-276) and detected in 56% patients (median, 7%; range, 0-60%), at week 12, evaluated in 13 patients (median, 11; range, 0-449) and detected in 62% patients (median, 15%; range, 0-88%), and at week 24, evaluated in 11 patients (median, 8; range, 0-35) and detected in 55% patients (median, 26%; range, 0-92%). Significant decrease in the ratio of PD-L1 positive CTCs between week 4 to week 8 was determined by Wilcoxon-matched-pairs signed rank test. Cutoff value of PD-L1-positivity rates in CTCs to segregate durable clinical benefit (DCB) from non-DCB was calculated to be 7% at week 8 according to ROC curve. Progression free survival was significantly longer in the patients with 7% or more of PD-L1 positivity rates (n = 8) than in those with less than 7% (n = 8) (p < 0.01) at week 8, suggesting the predictive significance of early evaluation of PD-L1 expression on CTCs after nivolumab treatment. Interestingly, CTC count in 7 out of 9 patients who had a partial response (PR) transiently increased from the one immediately prior to achieving a PR. Progression free survival was significantly longer in patients with 1.5 times or more increase of CTC count (n = 6) than in those with less than 1.5 times (n = 3) (p < 0.05), suggesting the transient tumor cell intravasation due to nivolumab treatment and its potential correlation with the duration of response depending on its magnitude. Conclusions: Longitudinal evaluation of PD-L1-expressing CTCs may have a predictive potential in NSCLC patients treated with nivolumab. Citation Format: Yasuhiro Koh, Mio Ikeda, Shunsuke Teraoka, Koichi Sato, Nahomi Tokudome, Atsushi Hayata, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Katsuya Endo, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Longitudinal investigation of PD-L1 expression on circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 775.

Purpose: Though programmed death receptor-ligand 1 (PD-L1) expression on tumor tissue is a validated predictive biomarker for PD-1 pathway blockade in NSCLC, its longitudinal change during the treatment remains elusive. We have previously reported that PD-L1-positive rate on CTCs at baseline was correlated with response to nivolumab. Here, we conducted a longitudinal investigation of PD-L1 expression on CTCs in NSCLC patients treated with nivolumab and its association with clinical outcome. Methods: For CTC detection, 3mL of peripheral blood was collected from advanced NSCLC patients treated with nivolumab at baseline and week 4, 8, 12, and 24 or until progressive disease (PD). The samples were enriched using the microcavity array system based on the difference in size. CTCs were defined as DAPI-positive, CK-positive, and CD45-negative cells and evaluated for PD-L1 expression by additional immunostaining. Results: Forty-five patients were enrolled between January 2016 and January 2018 at Wakayama Medical University. The patient characteristics were as follows: median age 68 years (range, 49-86); male 75%; stage III/IV, 25/75%; adenocarcinoma/ squamous cell carcinoma/other, 68/25/7%; partial response/stable disease/PD/not evaluable, 20/25/45/9%. At baseline, CTCs were evaluated in 44 patients (median, 13; range, 1-104) and PD-L1 positive CTCs were detected in 82% of patients (median 29%; range, 0-100%); at week 4, evaluated in 31 patients (median, 4; range, 0-115) and detected in 58% of patients (median, 13%; range, 0-100%); at week 8, evaluated in 16 patients (median, 11; range, 1-276) and detected in 56% of patients (median, 7%; range, 0-60%); at week 12, evaluated in 13 patients (median, 11; range, 0-449) and detected in 62% of patients (median, 15%; range, 0-88%); and at week 24, evaluated in 11 patients (median, 8; range, 0-35) and detected in 55% of patients (median, 26%; range, 0-92%). Cutoff value of PD-L1-positivity rates in CTCs to segregate durable clinical benefit (DCB) from non-DCB was calculated to be 7% at week 8 according to receiver operating characteristic curve. Progression-free survival was significantly longer in the patients with less than 7% of PD-L1 positivity rates (n = 8) than in those with 7% or more (n = 8) (p < 0.01) at week 8, suggesting the predictive significance of early evaluation of PD-L1 expression on CTCs after nivolumab treatment. It, however, did not translate into the predictive significance in overall survival. The change in CTC count at PD did not significantly differ between PD and last evaluation point before PD compared to that in non-PD patients at each evaluation point, suggesting the change in CTC count may not be predictive of disease progression upon nivolumab treatment. Conclusion: Longitudinal evaluation of PD-L1-expressing CTCs may have a predictive potential in NSCLC patients treated with nivolumab. Evaluation in a larger cohort is warranted to confirm the results. Citation Format: Mio Ikeda, Yasuhiro Koh, Shunsuke Teraoka, Koichi Sato, Nahomi Tokudome, Atsushi Hayata, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Katsuya Endo, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Longitudinal evaluation of PD-L1 expression on circulating tumor cells (CTCs) in non-small cell lung cancer (NSCLC) patients treated with nivolumab [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B27.

Background Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles. Results We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images. Conclusions Our protocol should be adaptable for monitoring activation for other plant small GTPases and protein–protein interactions for other FRET sensors in various plant cells.

Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles. We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in rice cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts withRaichu-OsRac1and acquisition of FRET images. Our protocol should be widely adaptable for monitoring activation for other plant small GTPases and for other FRET sensors in various plant cells.

Circulating tumor cells (CTCs), defined as tumor cells circulating in the peripheral blood of patients with solid tumors, are relatively rare. Diagnosis using CTCs is expected to help in the decision-making for precision cancer medicine. We have developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Herein, we evaluated the system using blood samples from non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC) patients. To evaluate the recovery of CTCs, preclinical experiments were performed by spiking NSCLC cell lines (NCI-H820, A549, NCI-H23 and NCI-H441) into peripheral whole blood samples from healthy volunteers. The recovery rates were 70% or more in all cell lines. For clinical evaluation, 6 mL of peripheral blood was collected from 50 patients with advanced lung cancer and from 10 healthy donors. Cells recovered on the filter were stained. We defined CTCs as DAPI-positive, cytokeratin-positive, and CD45-negative cells under the fluorescence microscope. The 50 lung cancer patients had a median age of 72 years (range, 48-85 years); 76% had NSCLC and 20% had SCLC, and 14% were at stage III disease whereas 86% were at stage IV. One or more CTCs were detected in 80% of the lung cancer patients (median 2.5). A comparison of the CellSearch system with our MCA system, using the samples from NSCLC patients, confirmed the superiority of our system (median CTC count, 0 versus 11 for CellSearch versus MCA; p = 0.0001, n = 17). The study results suggest that our MCA system has good clinical potential for diagnosing CTCs in lung cancer.

Purpose: Circulating tumor cells (CTCs) have a potential for the noninvasive decision-making. AXL, a receptor tyrosine kinase is associated with promoting cell proliferation, migration, epithelial-to-mesenchymal transition and drug resistance. We previously reported that the establishment of the detection method of AXL-expressing CTCs by an automated microcavity array (MCA) system incorporating mesenchymal marker vimentin (VM). Here we analyzed the clinicopathological correlation with VM-positive AXL-expressing CTCs in advanced non-small-cell lung cancer (NSCLC) patients. Methods: For detecting CTCs, 6 mL of peripheral blood collected from patients was divided into two portions and used for CTC identification in parallel incorporating markers cytokeratin (CK) and VM, respectively. An automated MCA system was used for enrichment and staining for CD45, DAPI, CK or VM with the additional staining for AXL. DAPI-positive, CK or VM-positive, and CD45-negative cells were defined as CTCs. All statistical analyses were carried out using GraphPad Prism 6 and Mann-Whitney U test, Fisher’s exact test, and rog-rank test were performed accordingly. A p value Citation Format: Mio Ikeda, Yasuhiro Koh, Shunsuke Teraoka, Koichi Sato, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Katsuya Endo, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Clinical significance of vimentin-positive AXL-expressing circulating tumor cells in advanced non-small-cell lung cancer patients [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A032. doi:10.1158/1535-7163.TARG-19-A032

Purpose: Noninvasive diagnostics has been developed over the last decade and we have previously reported that CTCs can be utilized for evaluating molecular features of NSCLC. AXL, a receptor tyrosine kinase is linked to epithelial-to-mesenchymal transition (EMT) leading to cancer progression and regarded as a potential therapeutic target. However, many of current technologies rely on epithelial markers to detect CTCs and that makes it difficult to detect AXL-expressing CTCs. Here, we established the detection of AXL expression on CTCs using MCA system. Methods: Preclinical experiments were performed using NSCLC cell lines H1299, PC9, and HCC827 and a breast cancer cell line MDA-MB231 with varying cytokeratin (CK) and AXL expression levels. The cells were spiked into 3 ml of peripheral blood from healthy donors, then enriched using MCA system, and detected by staining for CD45, DAPI, CK or vimentin (VM) with the addition staining for AXL. For clinical evaluation, 3ml of peripheral blood was collected from advanced NSCLC patients. Results: DAPI-positive, CK or VM-positive, and CD45-negative cells were defined as CTCs. In spike-in experiments, when CK was used as a marker, AXL expression was detected in 5 and 17% of high CK-expressing HCC827 and PC9 cells, respectively and detected in 52 and 75% of low CK-expressing H1299 and MDA-MB231 cells, respectively. On the other hand, when VM was used as a marker, AXL expression was detected in 72 and 88% of high VM-expressing MDA-MB231 and H1299 cells, respectively whereas detected in 1 and 7% of PC9 and HCC827 cells with low VM expression, respectively. Twenty-four patients were enrolled in the clinical study. The patient characteristics were as follows: median age 69.5 years (range, 49-84); male 88%; stage III/IV, 25/75%; adenocarcinoma/ squamous cell carcinoma/ other, 63/33/4%. Both CK and VM staining in 17 patients, only CK in 6 patients, and only VM in 1 patient were performed. CK-positive single CTCs were detected in all patients (median, 4; range, 1-50) and AXL-expressing CK-positive single CTCs were detected in 26% of patients (median, 0; range, 0-1). On the other hand, VM-positive single CTCs were detected in 94% of patients (median, 6.5; range, 0-128) and AXL-expressing VM-positive single CTCs were detected in 89% of patients (median, 1; range, 0-106). Significantly more AXL-expressing single CTCs were detected in VM-positive than CK-positive (p < 0.001). Notably, all of identified CTC clusters were positive for only VM, not CK and AXL-expressing CTC clusters were detected in 33% of patients (median, 0; range, 0-22). Conclusion: Our data indicate that incorporating VM staining is necessary to detect AXL-positive CTCs due to EMT. Further clinical evaluation is warranted for validating the method and the clinical significance of AXL-positive CTCs. Citation Format: Mio Ikeda, Yasuhiro Koh, Shunsuke Teraoka, Jun Oyanagi, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Hiroaki Akamatsu, Yuichi Ozawa, Keiichiro Akamatsu, Masayuki Higuchi, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Detection of AXL-expressing circulating tumor cells (CTCs) in non-small-cell lung cancer (NSCLC) patients using an automated microcavity array (MCA) system [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 414.

Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gβ mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.

Background: Anti-PD-1 antibody nivolumab has become a new standard treatment for pretreated, advanced non-small cell lung cancer (NSCLC). Although PD-L1 expression on tumor tissue has a predictive value, significance of its expression on circulating tumor cells (CTCs) is unknown. Here, we conducted a sequential evaluation of PD-1-expressing CTCs in NSCLC patients treated with nivolumab. Methods: Advanced NSCLC patients who received nivolumab at Wakayama Medical University Hospital were enrolled in the study (UMIN000024414). Nivolumab was administered 3 mg/kg bi-weekly until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected in an EDTA collection tube (BD vacutainer) and processed within 3 hours for CTC evaluation at baseline, week 4 and week 8. CTCs were detected using automated microcavity array system (Hitachi Chemical Co.). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs using anti-PD-L1 antibody, clone 28-8 (Abcam). Results: Thirty-eight patients were registered in the study between January 2016 and September 2016. Clinical characteristics of the patients were as follows: median age 68 (range, 49 to 86); male 73 %; stage IV 100 %; squamous/non-squamous, 30/65 %. Regarding nivolumab treatment, overall response rate (ORR) was 22% (95% confidence interval [CI]: 10-38%), and median progression-free survival (PFS) was 62 days (95%CI: 40-235 days). At baseline, CTCs were detected in all patients (median, 15; range, 1-90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs varied from 6% to 100%. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥50%. PD-L1 status on CTCs was not correlated with that on tumor tissues both using proportional score and H score (Spearman's correlation: r = 0.0007 and 0.08, respectively). On CTCs, patients with PD-L1 ≥50 % have significantly higher disease control rate than those with below 50% (83.3% versus 36.4%, p Conclusions: Sequential monitoring of PD-L1 expression on CTCs during nivolumab treatment was successfully conducted. PD-L1 expression on CTCs at baseline was a strong predictor in efficacy. Predictive significance of PD-L1-positive CTCs should be evaluated in a larger validation cohort. Citation Format: Yasuhiro Koh, Hiroaki Akamatsu, Keita Mori, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Masayuki Higuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Predictive impact of sequential evaluation of PD-L1-expressing circulating tumor cells in NSCLC patients treated with nivolumab [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 5595.

Background: Nivolumab (anti-PD-1 antibody) has become a new standard treatment in pretreated, advanced non-small cell lung cancer (NSCLC). Although strong expression of PD-L1 on tumor tissue has predictive value, significance of its expression on circulating tumor cell (CTC) is unknown and its status can be changed during the treatment. Here, we conducted a serial evaluation of PD-1-expressing CTCs in NSCLC patients treated with nivolumab. Methods: Advanced NSCLC patients who receive nivolumab at Wakayama Medical University Hospital were enrolled in this prospective observational study (registered at UMIN (000024414)). Nivolumab was administered 3 mg/kg biweekly until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected in a EDTA collection tube (BD vacutainer) and processed within 3 hours for CTC evaluation at baseline, week 4, and week 8. CTCs were detected using microcavity array system (Hitachi Chemical Co.). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs using anti-PD-L1 antibody, clone 28-8 (Abcam). Results: Thirty-eight patients were registered in this study between January 2016 and September 2016. Clinical characteristics of the patients were as follows: median age 68 (range, 49 to 86); male 73%; stage IV 100%; squamous/non-squamous, 30/65%. Regarding nivolumab treatment, overall response rate (ORR) was 22% (95% confidence interval (CI): 10-38%), and median progression-free survival (PFS) was 62 days (95%CI: 40-235 days). At baseline, CTCs were detected in all patients (median, 15; range, 1-90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs varied from 6% to 100%. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥ 50%. PD-L1 status on CTCs was not correlated with that on tumor tissues both using proportional score and H score (Spearman’s correlation: r = 0.0007 and 0.08, respectively). On CTCs, patients with PD-L1 ≥ 50 have significantly higher disease control rate than those with below 50% (83.3% versus 36.4%, p Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Keita Mori, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Masayuki Higuchi, HIsashige Kanbara, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Sequential tracking of PD-L1 expression on circulating tumor cells in NSCLC patients treated with nivolumab [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A056.

Energy transfer and shielding effect are studied in wire-type dendrimers (G n PPV: n th generation dendrimer with poly-( p -phenylenevinylene) backbone; n =1, 2) having oligophenylene light-harvesting (LH) antenna. Following the excitation of the LH-antennae, backbone-polymers in G n PPV give rise to intense photoluminescence (PL) bands. This is due to the presence of highly efficient energy transfer from the LH-antennae to the backbone-polymers. The intensity of backbone-PL increases faster than the decay of the antenna-PL. This result indicates that rapid energy transfer from antenna to backbone takes place utilizing the overlap of wavefunctions in the excited states. In G2PPV having larger LH-antenna, shielding effect against inter-backbone interactions is recognized more effectively than G1PPV. In solid films of G n PPV, red shifts of the backbone-PL bands are observed. This is caused by inter-backbone interactions of the wire-type dendrimers due to aggregation. The extent of the red shift in G2PPV is smaller than that of G1PPV. This result suggests that the larger LH-antenna in G2PPV substantially wraps its backbone-polymer and shields the inter-backbone interactions.

Alkamides and N-acilethanolamides are a class of lipid compounds related to animal endocannabinoids of wide distribution in plants. We investigated the structural features required for alkamides to regulate plant development by comparing the root responses of Arabidopsis (Arabidopsis thaliana) seedlings to a range of natural and synthetic compounds. The length of the acyl chain and the amide moiety were found to play a crucial role in their biological activity. From the different compounds tested, N-isobutyl decanamide, a small saturated alkamide, was found to be the most active in regulating primary root growth and lateral root formation. Proliferative-promoting activity of alkamide treatment was evidenced by formation of callus-like structures in primary roots, ectopic blades along petioles of rosette leaves, and disorganized tumorous tissue originating from the leaf lamina. Ectopic organ formation by N-isobutyl decanamide treatment was related to altered expression of the cell division marker CycB1:uidA and an enhanced expression of the cytokinin-inducible marker ARR5:uidA both in roots and in shoots. The involvement of cytokinins in mediating the observed activity of alkamides was tested using Arabidopsis mutants lacking one, two, or three of the putative cytokinin receptors CRE1, AHK2, and AHK3. The triple cytokinin receptor mutant was insensitive to N-isobutyl decanamide treatment, showing absence of callus-like structures in roots, the lack of lateral root proliferation, and absence of ectopic outgrowths in leaves under elevated levels of this alkamide. Taken together our results suggest that alkamides and N-acylethanolamides may belong to a class of endogenous signaling compounds that interact with a cytokinin-signaling pathway to control meristematic activity and differentiation processes during plant development.

Since their discovery as cell-division factors in plant tissue culture about five decades ago, cytokinins have been hypothesized to play a central role in the regulation of cell division and differentiation in plants. To test this hypothesis in planta , we isolated Arabidopsis plants lacking one, two, or three of the genes encoding a subfamily of histidine kinases ( CRE1 , AHK2 , and AHK3 ) that function as cytokinin receptors. Seeds were obtained for homozygous plants containing mutations in all seven genotypes, namely single, double, and triple mutants, and the responses of germinated seedlings in various cytokinin assays were compared. Both redundant and specific functions for the three different cytokinin receptors were observed. Plants carrying mutations in all three genes did not show cytokinin responses, including inhibition of root elongation, inhibition of root formation, cell proliferation in and greening of calli, and induction of cytokinin primary-response genes. The triple mutants were small and infertile, with a reduction in meristem size and activity, yet they possessed basic organs: roots, stems, and leaves. These results confirm that cytokinins are a pivotal class of plant growth regulators but provide no evidence that cytokinins are required for the processes of gametogenesis and embryogenesis.

We investigated 160 kinds of pesticide residues in 715 samples of 116 kinds of foods distributed in Kitakyushu city. Sixty kinds of pesticides were detected in 55 kinds of foods (204 samples) in the range of 0.002-22 mg/kg. Five kinds of pesticides in 7 samples violated the residue standards and the indication of "unused". The detection ratios of unregulated pesticide in domestic and imported foods were 27.8 and 33.0%, respectively. Iprodione, dicofol, diethofencarb, procymidone and chlorfenapyr (for domestic food) and total bromine, benomyl, chlorpyrifos, dicofol, fenvalerate, cypermethrin and dimethoate (for imported food) showed relatively high detection ratios. Chinese cabbage, garland chrysanthemum, tomatoes and green teas (domestic) and broccoli, bananas, grapefruit, lemons, oranges, frozen edamame and frozen kidney beans (imported) showed high relative pesticide detection ratios. Residual pesticides were detected with relatively high frequency in imported fruits, imported frozen foods and imported processed foods.

Background: Blockade of programmed death receptor-1 (PD-1) pathway is effective against solid tumors including lung cancer. Although PD-ligand 1 (PD-L1) expression on tumor tissue is expected as a potent predictive biomarker, its detection remains challenging due to its dynamic and unstable status. Circulating tumor cells (CTCs) have potential as an alternative material for non-invasive and real-time diagnosis. Here, we evaluated the PD-L1 expression on CTCs in patients with lung cancer and investigated the agreement between tumor tissues and CTCs. Material and methods: CTCs were captured and immune-stained using microcavity array system. CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. PD-L1 expression on CTCs was evaluated by addition of the process of PD-L1 immunocytochemistry. For CTCs detection, 3 ml of peripheral whole blood was collected from the patients who consented in written form and PD-L1 immunohistochemistry was performed using corresponding tumor tissues. Results: Sixty-seven lung cancer patients were enrolled in the study between July 2015 and April 2016 at Wakayama Medical University. Patient characteristics were as follows: median age 71 (range, 39 to 86); male 72%; stage II-III/IV, 15/85%; non-small cell lung cancer (NSCLC)/small cell lung cancer (SCLC)/Other, 73/21/6%. CTCs were detected in 66 out of 67 patients (median 19; range, 0 to 115) and more than 5 CTCs were detected in 78% of patients. PD-L1-expressing CTCs were detected in 73% of patients and the proportion score (PS) of PD-L1-expressing CTCs ranged from 3% to 100%, suggesting intra-patient heterogeneity of PD-L1 expression on CTCs. Significantly more PD-L1-expressing CTCs were detected in patients without EGFR mutations than those with EGFR mutations (P = 0.0433). Tumor tissues were available from 28 patients and were immune-stained for PD-L1. Seven showed the PS of PD-L1-expressing tumor cells < 1%, 11 showed 1-49%, and 10 showed ≥ 50%. No positive correlation was observed on PD-L1 expression between tumor tissues and CTCs based on PS (R2 = 0.0034). Three adenocarcinoma cases with PD-L1-positive tumor tissue did not harbor any PD-L1-expressing CTCs and conversely, three adenocarcinoma cases with PD-L1-negative tumor tissue harbored PD-L1-expressing CTCs, showing the discrepancy between tumor tissues and CTCs. It is also noteworthy that SCLC patients had perfect agreement on PD-L1 expression between tumor tissues and CTCs. Conclusions: PD-L1 expression was detectable on CTCs in lung cancer patients and intra-patient heterogeneity of its expression was observed. There was no agreement between tumor tissues and CTCs on PD-1 expression. Further investigation is warranted to better understand the clinical significance of PD-L1-expressing CTCs. Citation Format: Hiroaki Akamatsu, Yasuhiro Koh, Satomi Yagi, Satoshi Kambayashi, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Nahomi Tokudome, Keiichiro Akamatsu, Masayuki Higuchi, Hisashige Kanbara, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. PD-L1 expression on circulating tumor cells and its comparison with tumor tissues in Japanese lung cancer patients [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 3778. doi:10.1158/1538-7445.AM2017-3778

11541 Background: PD-L1 expression on tumor tissue is associated with response to PD-1 blockade in NSCLC. Here, we conducted a serial evaluation of PD-1-expressing circulating tumor cells (CTCs) as a potential real-time diagnostic modality in NSCLC patients treated with nivolumab. Methods: Advanced NSCLC patients after failure of at least one prior chemotherapy regimen received nivolumab monotherapy (3mg/kg, q2W) until progressive disease (PD) or unacceptable toxicity. Peripheral whole blood (3 mL) was collected for CTC evaluation at baseline and at week 4. CTCs were detected using microcavity array system (Hitachi Chemical Co., Ltd, Chikusei, Japan). PD-L1 expression was immunohistochemically examined on both tumor tissues and CTCs. This study was registered at UMIN (ID: 000024414). Results: Thirty patients were registered in the study between January 2016 and September 2016 at Wakayama Medical University Hospital and 29 were included in the analysis. Demographics of the patients were as follows: median age 70 (range, 49 to 86); male 73 %; stage IV, 100 %; squamous/non-squamous, 27/73 %. At baseline, CTCs were detected in all patients (median, 15; range, 1 to 90) and PD-L1-expressing CTCs were detected in 87% of patients. Tumor proportion score (TPS) of PD-L1 expression on CTCs ranged from 6% to 100%, indicating intrapatient heterogeneity. Matched tumor tissues were available from 14 patients and 7 showed the PD-L1 TPS ≥ 50%. No positive correlation was observed on PD-L1 expression between tumor tissues and CTCs based on TPS (R2 = 0.0035). Overall response rate was 25% (7/29), and disease control rate was 54% (15/29). Total CTC count was significantly decreased after nivolumab treatment at week 4 (p < 0.05), but no significant change was observed in PD-L1 TPS on CTC. Patients harboring CTCs with PD-L1 TPS 50% or more at baseline were significantly more likely to achieve non-PD than those harboring CTCs with TPS less than 50% (p < 0.05). Conclusions: This is the first report on a serial monitoring of PD-L1 expression on CTCs in patients treated with nivolumab. PD-L1-expressing CTCs are suggested to hold potential for predicting clinical benefit. Clinical trial information: 000024414.

L'A. retrace l'histoire du prefixe y- du moyen anglais, descendant du vieil anglais ge-. L'analyse des ecrits de Chaucer, ou l'on rencontre assez frequemment y-, permet de determiner les fonctions stylistique et grammaticale de ce prefixe

Background and purpose: Immune-checkpoint blockade with anti-programmed death-1 (PD-1) antibodies is rapidly emerging for the treatment of human malignancies including lung cancer. Although programmed death-ligand 1 (PD-L1) has been studied as a predictive biomarker, detection and evaluation of PD-L1 expression level on tissue samples remain challenging due to its dynamic and unstable expression. Thus the diagnostic tool for real-time monitoring of PD-L1 expression is critically needed. Here, we assessed the expression pattern of PD-L1 on circulating tumor cells (CTCs) by using microcavity array (MCA) system in patients with advanced non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Experimental procedure: PD-L1 staining on CTCs was established using NSCLC cell lines H820, H441, A549 and H23 expressing varying levels of PD-L1 spiked in the peripheral blood obtained from healthy donors. For clinical evaluation, 3 ml of peripheral whole blood was collected from 20 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Cells were captured and immuno-stained by using the automated MCA system (Hitachi Chemical Co., Ltd). CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. PD-L1 expression level on CTCs was visualized by addition of PD-L1 immunocytochemistry procedure. High-resolution fluorescent images were obtained using fluorescence microscope (Carl Zeiss Microscopy Co., Ltd). Results: Characteristics of 20 lung cancer patients enrolled in clinical study were as follows: median age 74 (range, 48 to 84); male 60%; stage III/IV, 10/90%; NSCLC/SCLC, 70/30%. More than 2 CTCs were identified in 14 patients (median 22.5; range, 4 to 71), and PD-L1 positive CTCs were detected in 12 patients (median 5; range, 2 to 15). No correlation was detected between the number of total CTCs and that of PD-L1 positive CTCs in each patient (R2 = 0.05). We found a total of 25 CTC clusters from 20 patients, of which PD-L1 expression was both homogenous and heterogeneous. It is noteworthy that clustered CTCs have larger proportion of PD-L1 positive CTCs per whole clustered CTCs than that of non-clustered CTCs (24/54, 44% versus 51/347, 15%, respectively). We further focused on CTC-interacting white blood cells, which intensively bound with aggregated CTCs rather than single CTC (12/54, 22% versus 43/337, 13%, respectively). Our data implicate that PD-L1 expression on CTC correlates with aggregation of CTCs (p < 0.05). Conclusions: Our results showed that PD-L1 expression on CTCs was detectable and there is intrapatient heterogeneity of its expression in patients with advanced lung cancer. Further investigation is warranted to better understand the biological importance of the correlation between PD-1 expression and CTC aggregation and CTC bound to white blood cells. Citation Format: Woong Kim, Yasuhiro Koh, Hiroaki Akamatsu, Satomi Yagi, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Differential expression of PD-L1 on circulating tumor cells among patients with advanced lung cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2257.

Background and Purpose: Circulating tumor cells (CTCs) are relatively rare cells defined as tumor cells circulating in the peripheral blood of patients with solid tumors. Diagnosis utilizing CTCs is expected to help guide decision-making for precision cancer medicine. We developed an automated microcavity array (MCA) system to detect CTCs based on the differences in size and deformability between tumor cells and normal blood cells. Here we evaluated its performance using preclinical spike-in model and blood samples from non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) patients. Material and method: The automated MCA system consists of components such as chambered cartridge containing micro metal filter, reagent and waste reservoirs, and peristaltic pump. To evaluate the recovery of CTCs, preclinical experiments using NSCLC cells, NCI-H820, A549, NCI-H441 and NCI-H23 spiked into peripheral whole blood from healthy volunteers were performed. For clinical evaluation, 6 mL of peripheral whole blood was collected from 50 advanced lung cancer patients prior to the initiation of chemotherapy and from 10 healthy donors. Samples were collected in an EDTA-containing tube and were processed within 3 hours of blood draw. Recovered cells on the filter were then fixed, permeabilized, and stained automatically and high-resolution fluorescent images were obtained using fluorescence microscope. We defined CTC as DAPI-positive, cytokeratin-positive and CD45-negative cell. Results: Results of the preclinical study showed that up to 90% of spiked-in tumor cells were recovered, confirming that the detection sensitivity by this automated device is on par with that by previous manual detection procedure. Demographics of 50 lung cancer patients enrolled in clinical study were as follows: median age 72 (range, 48 to 85); male 66%; stage III/IV 12/88%; NSCLC/SCLC 78/22%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 / 6 mL, respectively. Three or more CTCs were detected in 71% of patients with advanced lung cancer (39 out of 50) and five or more CTCs were detected in 52% of patients (26 out of 50) (median CTC count 13.5). Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those with intrathoracic metasitasis (median CTC count, 8 versus 4, p = 0.058). A head-to-head comparison between CellSearch system and our system was conducted in NSCLC patients, showing the superiority of our system (median CTC count, 0 versus 11.25, p = 0.0001, n = 17). Conclusions Our results suggest that the automated MCA device has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This device also enables higher throughput owing to its automated procedure. Further clinical evaluation including the detection of PD-L1 expression will be performed in an expansion cohort. Citation Format: Satomi Yagi, Yasuhiro Koh, Hiroaki Akamatsu, Woong Kim, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Hiroki Ueda, Nobuyuki Yamamoto. Development of an automated device for size-based enrichment and isolation of circulating tumor cells in lung cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2244.

e23036Background: Immune-checkpoint blockage with anti-PD-1 antibodies is effective against human malignancies including lung cancer. Although the expression of PD-L1 on tumor cells is expected as ...

The countermeasures against toxic substances or musty odor cause substances in the source for water supply have become a serious subject with the pollution of environmental waters (Coleman et al., 1980; Nakasugi, 1993; Tanada et al., 1995; U. S. EPA, 1993). For the purpose of the utilization of the photodecomposition method for the water purification at a water purification plant or a water purifier, we have investigated on the photodecomposition behaviors of organic pollutants (Matsuda et al., 2002; Okumura et al., 2003), total organic compounds (Tanizaki et al., 2005), formaldehyde (Tanizaki et al., 2005), total organic halides (Tanizaki et al., 2005), trihalomethanes (Tanizaki et al., 1997, 2005), geosmin (Okumura et al., 2004) and 2-methyl-iso-borneol (Okumura et al., 2004) using a titanium dioxide (TiO2) photocatalyst. Relatively high photodecomposition efficiencies were obtained for these substances. Pesticide is one of the toxic chemical substances polluting the source for water supply (Tanada et al., 1995), vegetables, fruits, etc (Ishikawa et al., 2004). Then, the residual pesticide in many kinds of food have already been regulated and the aiming standard values of 101 types of pesticide for tap water quality control were set up in 2003 (Ando, 2004). However, only 21 in 117 types of pesticide showed over 80% of removal ratio in the coagulation and sedimentation usually performed at a water purification plant (Ishikawa et al., 2006). Then, we investigated on the photodecomposition behaviors of the 5 types of pesticide which were largely used in Kitakyushu district. We have ever used silica gel having high decomposition efficiency as a carrier of TiO2 photocatalyst. However, a silica gel carrier was fragile. In addition, sufficient endurance of the carrier is demanded when the decomposition of chemicals in water and food or that disinfection using supersonic wave together is performed (Ishikawa et al., 2008). Then, we also investigated on the photodecomposition ability with an alumina carrier, which could stand against ultra violet (UV) light and water, instead of silica gel carrier.

The cytokinin class of plant hormones is perceived by transmembrane His-kinases (His-kinases) of the two-component system, otherwise known as the His-Asp phosphorelay system. When cytokinin receptors perceive cytokinins, they are autophosphorylated at a conserved His residue. The phosphoryl group is then transferred to downstream components of the His-Asp phosphorelay system. When the gene for a cytokinin receptor is introduced into yeast or Escherichia coli, the corresponding receptor feeds the phosphoryl group to the phosphorelay system of the host, in a cytokinin-dependent manner. Therefore, these microorganisms can be used as convenient cytokinin sensors, and can also be used to understand the properties of cytokinin-receptors. Furthermore, they may be used to screen for cytokinin agonists and antagonists, which would potentially be useful to regulate the growth of crops.

The cytokinin class of plant hormones is perceived by transmembrane His-kinases (His-kinases) of the two-component system, otherwise known as the His-Asp phosphorelay system. When cytokinin receptors perceive cytokinins, they are autophosphorylated at a conserved His residue. The phosphoryl group is then transferred to downstream components of the His-Asp phosphorelay system. When the gene for a cytokinin receptor is introduced into yeast or Escherichia coli, the corresponding receptor feeds the phosphoryl group to the phosphorelay system of the host, in a cytokinin-dependent manner. Therefore, these microorganisms can be used as convenient cytokinin sensors, and can also be used to understand the properties of cytokinin-receptors. Furthermore, they may be used to screen for cytokinin agonists and antagonists, which would potentially be useful to regulate the growth of crops.

Even though in recent years research and development of humanoid robots has increased, the major topics of research generally focus on how to make a robot perform specific motions such as walking. However, walking is only one of the complicated motions humans can perform. For robots to play an active role in society as our partner, they must be able to simulate precisely various kinds of human actions . We chose tai-chi as an example of complicated human actions and succeeded in programming a robot to perform the 24 fundamental tai-chi actions.

Circulating tumor cells (CTCs) are associated with prognosis of patients with advanced solid tumors including lung cancer and reflect characteristics of the respective primary tumor and its metastatic deposits. Assessment of CTCs is expected to improve effectiveness of anticancer therapy and to sophisticate our knowledge to cancer metastasis. We previously reported that detection of CTCs using microcavity array (MCA) system yielded the superior sensitivity than FDA-approved CellSearch system in patients with non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Here we developed the automated CTCs detection device and evaluated its performance using preclinical spike-in model and blood samples from NSCLC and SCLC patients. The feasibility study on the assessement of PD-L1 expression level was also performed. To evaluate the recovery of CTCs preclinically, NSCLC cells, H1975, A549, H441 and PC-14 were spiked into 6 mL of peripheral whole blood obtained from healthy volunteers. Then, cells were captured and immuno-stained by using automated MCA system. CTCs were defined as those positive for DAPI and cytokeratin (CK) and negative for CD45. Additionally, normal blood cells and cancer cells were distinguished according to their size. For clinical evaluation, 6 mL of peripheral whole blood was collected from 10 healthy donors and 30 advanced lung cancer patients prior to the initiation of chemotherapy. Head-to-head comparison with CellSearch system was also conducted. PD-L1 immunostaining was established in a preclinical spike-in study using NSCLC cell lines, H820, A549, H441, and H23 with varying PD-L1 expression levels and tested as an exploratory objective in a subset of patients enrolled in a clinical study. We confirmed that up to 90% of spiked-in tumor cells were recovered by the automated MCA system, suggesting that the detection sensitivity by this automated device is on par with that by previous detection procedure. Characteristics of 30 lung cancer patients in clinical study were as follows: median age 71 (range, 48 to 85); male 70%; stage III/IV 17/83%; NSCLC/SCLC 83/17%. Cells defined as CTC were detected in 2 cases out of 10 healthy volunteers, of which CTC count was 1 and 2 in 6 mL of peripheral blood, respectively. More than 2 CTCs were detected in 77% of patients with advanced lung cancer (n = 23/30) and more than 5 CTCs were detected in 50% of patients (n = 15/30) (median CTC count 5.5). Significantly more CTCs were detected by the automated MCA system than by CellSearch system. Among stage IV NSCLC patients, patients with extrathoracic metastasis tend to have more CTCs than in those without one (median CTC count, 8 versus 4, p = 0.058). No difference in CTC counts between NSCLC and SCLC was observed in this study cohort. PD-L1 expression was assessed in a subset of patients and intra-patient heterogeneity of PD-L1 staining among CTCs was observed in patients who harbor PD-L1-positive CTCs. Our results suggest that the automated MCA device for size-based enrichment and isolation of CTCs has a clinical potential for CTCs diagnosis towards precision medicine in lung cancer. This also enables us to deal with more samples owing to its automated procedure and higher throughput. Further clinical evaluation including PD-L1 expression will be performed in an expansion cohort and the biology of CTCs will be investigated utilizing this device. Citation Format: Woong Kim, Yasuhiro Koh, Hiroaki Akamatsu, Satomi Yagi, Ayaka Tanaka, Kuninobu Kanai, Atsushi Hayata, Ryota Shibaki, Masayuki Higuchi, Hisashige Kanbara, Takashi Kikuchi, Keiichiro Akamatsu, Masanori Nakanishi, Nobuyuki Yamamoto. Evaluation of a novel automated device for size-based enrichment and isolation of CTCs in patients with advanced lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B138.

The cell lineages that form the transporting tissues (xylem and phloem) and the intervening pluripotent procambial tissue originate from stem cells near the root tip. We demonstrate that in Arabidopsis, cytokinin phytohormones negatively regulate protoxylem specification. AHP6, an inhibitory pseudophosphotransfer protein, counteracts cytokinin signaling, allowing protoxylem formation. Conversely, cytokinin signaling negatively regulates the spatial domain of AHP6 expression. Thus, by controlling the identity of cell lineages, the reciprocal interaction of cytokinin signaling and its spatially specific modulator regulates proliferation and differentiation of cell lineages during vascular development, demonstrating a previously unrecognized regulatory circuit underlying meristem organization.

Daily intakes of 12 phenols which are possible endocrine disruptors were estimated in hospital meals from 2000 to 2001. 4-Nonylphenol (4-NP (mix)) and bisphenol A (BPA) were detected at levels of 5.0 to 19.4 ng/g and 0.2 to 1.1 ng/g, respectively. 4-tert-Butylphenol, 4-n-pentylphenol, 4 -tert-pentylphenol, 4-n-hexylphenol, 4-n-heptylphenol and 4-tert-octylphenol were detected at levels of 0.1 to 2.4 microg/g. The daily intakes of 4-NP (mix) and BPA were 5.8 microg/day and 0.42 microg/day, respectively. The daily intakes of other phenols were less than 1 microg/day.

A simultaneous determination method of 7 N-methylcarbamate and 7 urea pesticides in agricultural products by liquid chromatography/mass spectrometry (LC/MS) has been developed. Under reversed-phase liquid chromatographic conditions, 14 pesticides were analyzed using electrospray ionization (ESI) with simultaneous acquisition of positive ions and negative ions. Fourteen pesticides were extracted with acetone, 10% NaCl solution was added, and the pesticides were re-extracted with dichloromethane. The extract was concentrated under reduced pressure, and dissolved in methanol. The detection limits of 14 pesticides ranged from 0.0012 to 0.0056 microgram/g. The recoveries of pesticides were from 36.5 to 112.5% [RSD (n = 3) ranged from 0.5 to 48.1%] for 4 agricultural products.

Cytokinins are a class of plant hormones that are central to the regulation of cell division and differentiation in plants. It has been proposed that they are detected by a two-component system, because overexpression of the histidine kinase gene CKI1 induces typical cytokinin responses and genes for a set of response regulators of two-component systems can be induced by cytokinins. Two-component systems use a histidine kinase as an environmental sensor and rely on a phosphorelay for signal transduction. They are common in microorganisms, and are also emerging as important signal detection routes in plants. Here we report the identification of a cytokinin receptor. We identified Arabidopsis cre1 (cytokinin response 1) mutants, which exhibited reduced responses to cytokinins. The mutated gene CRE1 encodes a histidine kinase. CRE1 expression conferred a cytokinin-dependent growth phenotype on a yeast mutant that lacked the endogenous histidine kinase SLN1 (ref. 10), providing direct evidence that CRE1 is a cytokinin receptor. We also provide evidence that cytokinins can activate CRE1 to initiate phosphorelay signalling.

The porphyrin portion of cylodextrin(CD)-linked porphyrin derivatives (1)-(3) has been anchored onto a lipid bilayer and a light-induced intramolecular electron transfer between the porphyrin on (1) and (2) and the acceptor, α-naphthoquinone held in the CD cavity in aqueous solution, has been examined.

Summary The cytokinin class of plant hormones plays key roles in regulating diverse developmental and physiological processes [1]. Arabidopsis perceives cytokinins with three related and partially redundant receptor histidine kinases (HKs): CRE1 (the same protein as WOL and AHK4), AHK2, and AHK3 (CRE-family receptors) [2–5]. It is suggested that binding of cytokinins induces autophosphorylation of these HKs and subsequent transfer of the phosphoryl group to a histidine phosphotransfer protein (HPt) and then to a response regulator (RR), ultimately regulating downstream signaling events [6, 7]. Here we demonstrate that, in vitro and in a yeast system, CRE1 is not only a kinase that phosphorylates HPts in the presence of cytokinin but is also a phosphatase that dephosphorylates HPts in the absence of cytokinin. To explore the roles of these activities in planta, we replaced CRE1 with mutant versions of the gene or with AHK2 . Replacing CRE1 with CRE1(T278I), which lacks cytokinin binding activity [8] and is locked in the phosphatase form, decreased cytokinin sensitivity. Conversely, replacing CRE1 with AHK2, which favors kinase activity, increased cytokinin sensitivity. These results indicate that in the presence of cytokinins, cytokinin receptors feed phosphate to phosphorelay-integrating HPt proteins. In the absence of cytokinins, CRE1 removes phosphate from HPt proteins, decreasing the system phosphoload.

6-Trimethylsilylmethyl-5,6-dihydro-4H-1,2-oxazines, which are prepared by the reaction of α-halogeno-oximes with allylsilanes in the presence of anhydrous sodium carbonate, are converted into δ,Iµ-unsaturated-α-ketocarboxylic acid esters by the action of mineral acids.