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Genotyping of Plants by Combining Alkaline/SDS gDNA Extraction and CD-PCR CD-PCR was applied to the genotyping procedure of an I Arabidopsis thaliana i T-DNA tagline mutant (Fig. For both I mot1-1 i (a mutant T-DNA tagline of the molybdate transporter) and I abl2c i (a mutant T-DNA tagline of the anthocyanin-bluing transporter), the wild-type alleles and T-DNA-tagged mutant alleles were successfully identified by migration of allele-specific PCR products (Fig. The introduction of polymerase chain reaction (PCR) has dramatically simplified the gene determination process; however, preparing genomic DNA (gDNA) from test plants is still a time-consuming task. In a PCR that amplifies a 2-kb DNA fragment, -CD made a PCR containing at least 0.06% SDS possible (Supplementary Fig. [Extracted from the article]

Immune-checkpoint inhibitors (ICIs) have improved clinical outcomes and are becoming a standard treatment for many cancer types. However, these drugs also induce immune-related adverse events, among which interstitial lung disease (ILD) is potentially fatal. The underlying mechanism of ILD induction by ICIs is largely unknown. With the use of flow cytometry, we determined the expression levels of the immune-checkpoint proteins PD-1, TIM-3, TIGIT, LAG-3 and PD-L1 in T cells of bronchoalveolar lavage fluid (BALF) from patients with ICI-related ILD and compared them with those for patients with sarcoidosis or with ILD related to connective tissue disease or cytotoxic drug use. The proportions of CD8+ T cells positive for both PD-1 and TIM-3 or for TIGIT in BALF were significantly higher for ICI-related ILD patients than for those with other types of ILD. A prominent increase in the proportion of PD-1+PD-L1+ cells among CD8+ T cells was also apparent in BALF of a patient with a fatal case of ICI-related ILD, and the proportion of such cells was positively correlated with the grade of ICI-related ILD. Our data reveal the immune-checkpoint profiles of T cells in ICI-related ILD and may provide mechanistic insight into the development of this adverse event. [ABSTRACT FROM AUTHOR]

Copyright of Oncologist is the property of Oxford University Press / USA and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Proton-translocating inorganic pyrophosphatase (H+-PPase) actively translocates protons across membranes coupled with the hydrolysis of inorganic pyrophosphate (PPi). H+-PPase, which is composed of a single protein and uses a simple compound as a substrate, has been recognized as a new type of ion pump in addition to the P-, F- and V-type ion-translocating ATPases. H+- and Na+-PPases are distributed in various organisms including plants, parasitic protozoa, Archaebacteria and bacteria, but are not present in animals or yeast. Vacuolar H+-PPase has dual functions in plant cells: hydrolysis of cytosolic PPi to maintain the levels of PPi, and translocation of protons into vacuoles to maintain the acidity of the vacuolar lumen. Acidification performed with the vacuolar-type H+-ATPase and H+-PPase is essential to maintain acidic conditions, which are necessary for vacuolar hydrolytic enzymes and for supplying energy to secondary active transporters. Recent studies using loss-of-function mutant lines of H+-PPase and complementation lines with soluble PPases have emphasized the physiological importance of the scavenging role of PPi. An overview of the main features of H+-PPases present in the vacuolar membrane is provided in terms of tissue distribution in plants, intracellular localization, structure-function relationship, biochemical potential as a proton pump and functional stability. [ABSTRACT FROM AUTHOR]

OBJECTIVES This study intended to assess the clinicopathological features of the histological subtypes of adenocarcinoma of the lung in a large registry population. METHODS The Japanese Joint Committee of Lung Cancer Registry performed a nationwide retrospective registry study on the prognosis and clinicopathological profiles of 11 663 patients who underwent resection for primary lung neoplasm in 2004. The registry data of 7921 (62.5%) patients with adenocarcinoma were analysed regarding the prognosis and clinicopathological features according to the histological subtype of adenocarcinoma. The histological subtypes were defined according to the 1999 World Health Organization classification (third edition), where bronchioloalveolar carcinoma (BAC) is defined as adenocarcinoma with a pure bronchioloalveolar growth pattern without invasion. RESULTS The distribution of the histological subtype was acinar in 471 patients (7.5%), papillary in 2004 (32.2%), BAC in 1385 (22.3%), solid adenocarcinoma with mucin in 103 (1.7%) and adenocarcinoma with mixed subtypes (AMS) in 2257 (36.3%). The 5-year overall survival rates according to histological subtype were 63.4% for acinar, 72.9% for papillary, 90.3% for BAC, 54.4% for solid adenocarcinoma with mucin and 73.7% for AMS. While the survival rate in patients with BAC was significantly better than those for the other histological subtypes, acinar and solid adenocarcinoma with mucin had significantly worse prognoses than the other histological subtypes. The histological subtype was an independent predictor of survival in a multivariate analysis (P < 0.001). Regarding BAC, the pathological stage included not only Stage IA/IB (n = 1275; 92.1%), but also Stage II–IV (n = 110; 7.9%). One hundred twenty-five patients (9.0%) with BAC had recurrence, including both local and distant recurrence. CONCLUSIONS The histological subtype in adenocarcinoma significantly correlated with the prognosis. In BACs with recurrence or pathological stage II–IV, these tumours might have been classified as invasive adenocarcinoma rather than as BAC. The need for the rigorous pathological evaluation of adenocarcinomas that are considered to be a preinvasive or minimally invasive tumour should be addressed in the new lung adenocarcinoma classification to be proposed by the International Association for the Study of Lung Cancer, the American Thoracic Society and the European Respiratory Society. [ABSTRACT FROM PUBLISHER]

Cigarette smoke inhibits HDAC and reduces efferocytosis by alveolar macrophages.Efferocytosis, which is the homeostatic phagocytosis of apoptotic cells, prevents the release of toxic intracellular contents and subsequent tissue damage. Impairment of efferocytosis was reported in alveolar macrophages (AMs) of patients with chronic obstructive pulmonary disease (COPD), a common disease caused by smoking. In COPD, histone deacetylase (HDAC) activity is reduced in AMs. We investigated whether the reduction of HDAC activity is associated with the impairment of efferocytosis. Murine AMs were collected by bronchoalveolar lavage and their ability to efferocytose apoptotic human polymorphonuclear leukocytes was assessed. Pre-treatment of AMs with cigarette smoke extract (CSE) or trichostatin A (TSA), an HDAC inhibitor, suppressed efferocytosis and CSE reduced HDAC activity. TSA inhibited the activity of Rac, a key mediator of efferocytosis. These TSA-induced impairments were restored by treatment of AMs with aminophylline, a potent activator of HDAC. To further elucidate the underlying mechanism, we explored a role of CD9 in TSA-induced impairment of efferocytosis. CD9 is a transmembrane protein of the tetraspanin family that facilitates the uptake of several pathogens and other material. TSA profoundly down-regulated the expression of CD9 on AMs. The expression of CD9 was partly down-regulated by the Rac inhibitor. Pretreatment with an anti-CD9 mAb or CD9 small interfering RNA inhibited efferocytosis, which was attributable to the reduced binding of AMs to apoptotic cells. These results suggest that smoking impairs efferocytosis via inhibition of HDAC/Rac/CD9 pathways. Aminophylline/theophylline is effective in restoring the impairment of efferocytosis and might have benefit for the treatment of patients with COPD. [ABSTRACT FROM PUBLISHER]

The local distribution of both the vacuolar-type proton ATPase (V-ATPase) and the vacuolar-type proton pyrophosphatase (V-PPase), the main vacuolar proton pumps, was investigated in intact vacuoles isolated from Arabidopsis suspension-cultured cells. Fluorescent immunostaining showed that V-PPase was distributed evenly on the vacuolar membrane (VM), but V-ATPase localized to specific regions of the VM. We hypothesize that there may be membrane microdomains on the VM. To confirm this hypothesis, we prepared detergent-resistant membranes (DRMs) from the VM in accordance with well established conventional methods. Analyses of fatty acid composition suggested that DRMs had more saturated fatty acids compared with the whole VM in phosphatidylcholine and phosphatidylethanolamine. In the proteomic analyses of both DRMs and detergent-soluble mebranes (DSMs), we confirmed the different local distributions of V-ATPase and V-PPase. The observations of DRMs with an electron microscope supported the existence of different areas on the VM. Moreover, it was observed using total internal reflection fluorescent microscopy (TIRFM) that proton pumps were frequently immobilized at specific sites on the VM. In the proteomic analyses, we also found that many other vacuolar membrane proteins are distributed differently in DRMs and DSMs. Based on the results of this study, we discuss the possibility that VM microdomains might contribute to vacuolar dynamics. [ABSTRACT FROM PUBLISHER]

Most plants have two types of H+-translocating inorganic pyrophosphatases (H+-PPases), I and II, which differ in primary sequence and K+ dependence of enzyme function. Arabidopsis thaliana has three genes for H+-PPases: one for type I and two for type II. The type I H+-PPase requires K+ for maximal enzyme activity and functions together with H+-ATPase in vacuolar membranes. The physiological role of the type II enzyme, which does not require K+, is not clear. We focused on the type II enzymes (AtVHP2;1 and AtVHP2;2) of A. thaliana. Total amounts of AtVHP2s were quantified immunochemically using a specific antibody and determined to be 22 and 12 ng mg-1 of total protein in the microsomal fractions of suspension-cultured cells and young roots, respectively, and the values are approximately 0.1 and 0.2 %, respectively, of the vacuolar H+-PPase. In plants, AtVHP2s were detected immunochemically in all tissues except mature leaves, and were abundant in roots and flowers. The intracellular localization of AtVHP2s in suspension cells was determined by sucrose density gradient centrifugation and immunoblotting. Comparison with a number of marker proteins revealed localization in the Golgi apparatus and the trans-Golgi network. These results suggest that the type II H+-PPase functions as a proton pump in the Golgi and related vesicles in young tissues, although its content is very low compared with the type I enzyme. [ABSTRACT FROM AUTHOR]

H+-translocating pyrophosphatases (H+-PPases) are proton pumps that are found in many organisms, including plants, bacteria and protozoa. Streptomyces coelicolor is a soil bacterium that produces several useful antibiotics. Here we investigated the properties of the H+-PPase of S. coelicolor by expressing a synthetic DNA encoding the amino-acid sequence of the H+-PPase in Escherichia coli. The H+-PPase from E. coli membranes was active at a relatively high pH, stable up to 50°C, and sensitive to N-ethylmaleimide, N,N′-dicyclohexylcarbodiimide and acylspermidine. Enzyme activity increased by 60% in the presence of 120 mM K+, which was less than the stimulation observed with plant vacuolar H+-PPases (type I). Substitutions of Lys-507 in the Gly-Gln-x-x-(Ala/Lys)-Ala motif, which is thought to determine the K+ requirement of H+-PPases, did not alter its K+ dependence, suggesting that other residues control this feature of the S. coelicolor enzyme. The H+-PPase was detected during early growth and was present mainly on the plasma membrane and to a lesser extent on intracellular membranous structures. [ABSTRACT FROM PUBLISHER]

Despite the usefulness of the patch-clamp technique, its application to ion pumps and transporters in biomembranes is limited. We developed a novel method for determining the activity of a proton-pumping pyrophosphatase (H+-PPase) made of a single protein. We heterologously highly expressed the enzyme in Saccharomyces cerevisiae, prepared giant vacuoles from the cells, and measured a PPi-dependent electrical current of 18 pA (10.5 fA/µm2) using the patch-clamp technique in the whole-vacuole recording mode. We determined the inhibitor sensitivity and affinity for substrate (Km, 4.6 µM). The enzyme number in a giant vacuole (4.2 × 106) and the molecular activity of the expressed H+-PPase (14 s–1) were determined. An uncoupling-type H+-PPase mutant, of which the 263rd glutamate residue was replaced by aspartate, and of which H+ pump activity was not detected with the fluorescence quenching method, showed a weak current with a high Km. The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed. [ABSTRACT FROM PUBLISHER]

H+-pyrophosphatase (H+-PPase), which pumps H+ across membranes coupled with PPi hydrolysis, is found in most plants, and some parasitic protists, eubacteria and archaebacteria. We assayed a number of extracts derived from 145 marine invertebrates as to their inhibitory effect on plant vacuolar H+-PPase. Acylspermidine derivatives [RCONH(CH2)3N(CH3)(CH2)4N(CH3)2] from a soft coral (Sinularia sp.) inhibited the PPi-hydrolysis activity of purified H+-PPase and the PPi-dependent H+ pump activity (half inhibition concentration, 1 µM) of vacuolar membranes of mung bean. The apparent Ki was determined to be 0.9 µM. Acylspermidines did not affect the activity of vacuolar H+-ATPase, plasma membrane H+-ATPase, mitochondrial ATPase or cytosolic PPase. Acylspermidines inhibited the acidification of vacuoles in protoplasts, as found on monitoring by the acridine orange fluorescent method. These results indicate that acylspermidine derivatives represent new inhibitors of H+-PPase with relatively high specificity. [ABSTRACT FROM PUBLISHER]

To elucidate a relationship between lung cancer and tumor induction of environmental chemicals, the presence of 1-nitropyrene (1-NP) and benzo[]pyrene (B[]P) in 137 Japanese (97 male and 40 female), and 21 Chinese lung specimens was examined by HPLC, and GC–MS and environmental exposure discussed. Mortality due to lung cancer in Fuyuan County, China, is much higher than that of other cancers. We investigated 21 patients who were residents of Fuyuan County. All were female aged 28–64 years and were non-smoking farmers and cooks. The histological features of the tumors were adenocarcinoma, squamous cell carcinoma and small cell carcinoma. The incidence of cancer was due to inhaling soot from the combustion of coal used for cooking and indoor heating. The polycyclic aromatic hydrocarbons, chrysene, benzo[]fluor-anthene, B[]P, benzo[]perylene and pyrene, but not their nitrated derivatives, were detected in substantial amounts in the resected lungs. These mutagens and carcinogens normally originate in combustion products of coal, and are discussed as the possible initiators of the tumors in the lungs of these patients. All the Japanese lung specimens were also obtained from non-smokers. The concentrations of 1-NP averaged 21.3 ± 12.4 and 5.9 ± 2.4 pg/g of Japanese and Chinese samples respectively. The concentrations of B[]P averaged 180.2 ± 103.7 and 608.7 ± 477.1 pg/g of Japanese and Chinese samples respectively. Thus, Japanese and Chinese lung specimens were mainly contaminated by 1-NP and B[]P, respectively. Typical tissues from a carcinomatous human lung were examined. The patient was a 64-year-old Japanese male non-smoker and farmer who had raised chickens over a period of 40 years. The histological features of the tumor were those of keratinizing squamous cell carcinoma. 1-Nitropyrene, 1-nitro-3-hydroxypyrene, 1,3-dinitropyrene and chrysene were detected at concentrations of 0.11, 0.036, 0.095 and 0.16 ng respectively per gram of lung tissue. This cancer was due to long-term exposure to the combustion products of heavy oil used in a chicken house. [ABSTRACT FROM PUBLISHER]

Lessons Learned: Updated survival data for a phase I/II study of carboplatin plus nab‐paclitaxel and concurrent radiotherapy were collected.In the group of 58 patients who were enrolled at 14 institutions in Japan, the median overall survival was not reached and the 2‐year overall survival rate was 66.1% (95% confidence interval, 52.1%–76.8%).Results reveal encouraging feasibility and activity for this regimen. Background: We report the updated survival data for a phase I/II study of carboplatin plus nab‐paclitaxel (nab‐P/C) and concurrent radiotherapy (CRT) in patients with locally advanced non‐small cell lung cancer (NSCLC). Methods: Individuals between 20 and 74 years of age with unresectable NSCLC of stage IIIA or IIIB and a performance status of 0 or 1 were eligible for the study. Patients received weekly nab‐paclitaxel at 50 mg/m2 for 6 weeks together with weekly carboplatin at an area under the curve (AUC) of 2 mg/ml/min and concurrent radiotherapy with 60 Gy in 30 fractions. This concurrent phase was followed by a consolidation phase consisting of two 3‐week cycles of nab‐paclitaxel (100 mg/m2 on days 1, 8, and 15) plus carboplatin (AUC of 6 on day 1). After the treatment, patients were observed off therapy. The primary endpoint of the phase II part of the study was progression‐free survival (PFS). Results: Between October 2014 and November 2016, 58 patients were enrolled at 14 institutions in Japan, with 56 of these individuals being evaluable for treatment efficacy and safety. At the median follow‐up time of 26.0 months (range, 4.0–49.6 months), the median overall survival (OS) was not reached (95% confidence interval [CI], 25.3 months to not reached) and the 2‐year OS rate was 66.1% (95% CI, 52.1%–76.8%). The median PFS was 11.8 months (95% CI, 8.2–21.0 months), and the 2‐year PFS rate was 35.9% (95% CI, 23.1%–48.9%). Subgroup analysis according to tumor histology or patient age revealed no differences in median PFS or OS. Long‐term follow‐up of toxicities did not identify new safety signals, and no treatment‐related deaths occurred during the study period. Conclusion: Concurrent chemoradiation with nab‐P/C was safe and provided a long‐term survival benefit for patients with locally advanced NSCLC. [ABSTRACT FROM AUTHOR]

Lessons Learned: Alectinib confers a pronounced survival benefit in patients with ALK rearrangement‐positive non‐small cell lung cancer and a poor performance status.Survival benefit of alectinib for patients with a poor performance status was consistent regardless of the presence of central nervous system metastases. Background: We previously reported a marked objective response rate (ORR) and safety for alectinib treatment in patients with ALK rearrangement‐positive non‐small cell lung cancer (NSCLC) and a poor performance status (PS) in the Lung Oncology Group in Kyushu (LOGiK) 1401 study. It remained unclear, however, whether alectinib might also confer a long‐term survival benefit in such patients. Methods: Eighteen patients with ALK rearrangement‐positive advanced NSCLC and a PS of 2, 3, or 4 (n = 12, 5, and 1, respectively) were enrolled in LOGiK1401 between September 2014 and December 2015 and received alectinib. We have now updated the survival data for the study. Results: The median follow‐up time for all patients was 27.3 months. The median progression‐free survival (PFS) was 16.2 months (95% confidence interval [CI], 7.1–30.8 months), and the median survival time (MST) and the 3‐year overall survival rate were 30.3 months (95% CI, 11.5 months to not reached) and 43.8% (95% CI, 20.8–64.7%), respectively. This survival benefit was similarly manifest in patients with a PS of 2 (MST, 20.5 months) and those with a PS of ≥3 (MST, not reached). PFS did not differ between patients with or without central nervous system (CNS) metastases at baseline (median of 17.5 and 16.2 months, respectively, p =.886). Conclusion: Alectinib showed a pronounced survival benefit for patients with ALK rearrangement‐positive NSCLC and a poor PS regardless of the presence of CNS metastases, a patient population for which chemotherapy is not indicated. [ABSTRACT FROM AUTHOR]

Lessons Learned: The biweekly GEM plus CBDCA dose and schedule showed satisfactory efficacy with mild toxicities in elderly patients with advanced NSCLC.The biweekly GEM plus CBDCA regimen could be considered an alternative to the 3‐week regimen in NSCLC. Background: The gemcitabine (GEM)‐carboplatin (CBDCA) combination is widely used for non‐small cell lung cancer (NSCLC) and has some efficacy in elderly patients; however, a high incidence of thrombocytopenia is observed, and the optimal dosage and administration schedules are unknown. This multicenter phase II trial evaluated the efficacy and tolerability of GEM‐CBDCA for elderly patients with chemotherapy‐naive NSCLC. Methods: Patients with chemotherapy‐naive performance status 0–1 and with stage IIIB/IV NSCLC were administered chemotherapy biweekly (GEM 1,000 mg/m2 with CBDCA area under the blood concentration‐time curve (AUC) 3 on days 1 and 15 every 4 weeks). The primary endpoint was the objective response rate (ORR), and the secondary endpoints were progression‐free survival (PFS), overall survival (OS), and safety. Results: Forty‐eight patients were enrolled. Median age was 76 years (range, 70–83); 35 patients were men (73%), and 27 patients had adenocarcinoma (56%). The ORR was 29.2% (95% confidence interval [CI], 17.0–44.1). The median PFS, median OS, and 1‐year survival was 5.9 months (95% CI, 4.1–6.6), 13.3 months (95% CI, 8.3–23.5), and 58%, respectively. Grade ≥3 hematological toxicities included neutropenia (29.2%), thrombocytopenia (4.2%), and anemia (20.8%). The incidence of grade ≥3 nonhematological toxicities was <5%. Conclusion: This GEM‐CBDCA combination administered biweekly showed satisfactory efficacy with mild toxicities in elderly patients with advanced NSCLC. [ABSTRACT FROM AUTHOR]

Copyright of Oncologist is the property of Oxford University Press / USA and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The function of the translation product of cDNA for Acetabularia vacuolar H+-pyrophosphatase was examined using the Saccharomyces cerevisiae VMA3-deficient strain. The open reading frame of Acetabularia H+-pyrophosphatase was revealed to encode 751 amino acids (721 or 751 amino acids in a previous paper). The acidification of the vacuole was observed by fluorescence microscopy when the cDNA was constructed in pYES2. Immunoblot analysis also supported the localization of the translation product in the vacuolar-membrane-enriched fraction. [ABSTRACT FROM AUTHOR]