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Although population-based cancer registries have reported lower incidence of Waldenstrӧm macroglobulinemia (WM) in East Asia than in Western countries, previous retrospective analyses have found the clinical features of WM to be similar in these two populations. To clarify the characteristics of Japanese WM patients, we retrospectively analyzed clinical and laboratory characteristics, treatments, outcomes, and prognostic factors in 93 patients with WM. Based on the Second International Workshop on WM (IWWM-2) criteria, symptomatic WM was found in 73 (78.5%) and asymptomatic WM in 20 (21.5%) of cases examined. The median overall survival (OS) was similar to that in reports from Western countries. Patients receiving treatment regimens including rituximab exhibited significantly better survival than those not given rituximab. Although prognostic factors for WM in Western countries may not apply to Japanese patients, our finding that newly diagnosed WM patients with pleural effusion have a poorer prognosis suggests that this may be a novel predictor of adverse prognosis in symptomatic WM.

We demonstrate that the copolymers containing boronic acid and pyrene units can be utilized for the fluorometric sensing of hydrogen peroxide (H 2 O 2 ) in aqueous solutions. The copolymer exists in a relatively extended conformation in the absence of H 2 O 2 , whereas the polymer chain is contracted by the reaction of boronic acid moieties with H 2 O 2 to form phenol groups. This conformational change induces aggregation of the originally isolated pyrene groups. As a result, relative intensity of excimer emission with respect to monomer emission increases with H 2 O 2 concentration. Accordingly, the present methodology enables us to measure H 2 O 2 by means of ratiometric fluorescence change in the range of 0-30  μ M.

Contrast-induced nephropathy (CIN) and chronic kidney disease (CKD) are associated with poor outcomes after primary percutaneous coronary intervention (PCI) for acute myocardial infarction (AMI); however, its combined prognostic significance remains unclear. We enrolled 577 patients with AMI undergoing primary PCI within 12 h after symptom onset and measured serum creatinine on admission and the next 3 days. CKD was defined as admission estimated glomerular filtration rate <60 ml/min/1.73 m 2 , and CIN was defined as creatinine increase ≥0.5 mg/dl or ≥25 % from baseline within the first 72 h. Patients were stratified according to the presence or absence of CKD and CIN. In patients with no CKD and no CIN (n = 244), no CKD but CIN (n = 152), CKD but no CIN (n = 127), and both CKD and CIN (n = 54), the 3-year major adverse cardiovascular events (MACE: a combination of all-cause mortality, nonfatal reinfarction, or heart failure requiring rehospitalization) were 8, 9, 13, and 35 %, respectively (p < 0.001). Multivariate analysis showed that as compared with no CKD and no CIN, hazard ratios (95 % CI) for MACE associated with no CKD but CIN, CKD but no CIN, and both CKD and CIN were 0.91 (0.44-1.84; p = 0.79), 1.11 (0.5-2.23; p = 0.77), and 2.98 (1.48-6.04; p = 0.002), respectively. In patients with AMI undergoing primary PCI, the combination of CKD and CIN is significantly associated with adverse long-term outcomes.

The compositions of the essential oils obtained from leaves and stems of Gynura bicolor DC. were analyzed by GC-MS. One hundred eight components of these oils were identified. (E)-β-caryophyllene (31.42 %), α-pinene (17.11 %), and bicyclogermacrene (8.09 %) were found to be the main components of the leaf oil, while α-pinene (61.42 %), β-pinene (14.39 %), and myrcene (5.10 %) were the major constituents of the stem oil. We found 73 previously unidentified components in these oils from G. bicolor. The oils were also subjected to odor evaluation. Eleven and 12 aroma-active compounds were detected in the leaf and stem oils, respectively. The abilities of these oils to inhibit acetylcholinesterase (AChE) activity were determined. The sesquiterpenoids in the oils were found to inhibit AChE activity more strongly than the monoterpenoids in the oils did. It was suggested that the three main components in each essential oil act synergistically against AChE activity. These results show that the essential oils obtained from G. bicolor are a good dietary source of AChE activity inhibition.

Background: In animal models of acute myocardial infarction (AMI), B-type natriuretic peptide (BNP) administered before and during coronary occlusion limits infarct size. However, the relation between plasma BNP levels and ischemia/reperfusion injury remains unclear., Methods: 302 patients with ST-segment elevation AMI (STEMI) received emergency percutaneous coronary intervention within six hours from the onset. The patients were divided into two groups according to the plasma BNP level before angiography: group L (n=151), BNP ≤ 32.2 pg/ml; group H (n=151), BNP >32.2 pg/ml. The Selvester QRS-scoring system was used to estimate infarct size., Results: The rate of ischemia/reperfusion injury immediately after reperfusion, defined as reperfusion ventricular arrhythmias (26% vs. 11%, p=0.001) and ST-segment re-elevation (44% vs. 22%, p=0.008), was higher in group L than in group H. Group L had a greater increase in the QRS score during percutaneous coronary intervention (3.55 ± 0.17 vs. 2.09 ± 0.17, p<0.001) and a higher QRS score 1 h after percutaneous coronary intervention (5.77 ± 0.28 vs. 4.51 ± 0.28, p=0.002). On multivariate analysis, plasma BNP levels in the lower 50th percentile were an independent predictor of reperfusion injury (odds ratio, 2.620; p<0.001). The odds ratios of reperfusion injury according to decreasing quartiles of BNP level, as compared with the highest quartile, were 1.536, 3.692 and 4.964, respectively (p trend=0.002)., Conclusions: Plasma BNP level before percutaneous coronary intervention may be a predictor of ischemia/reperfusion injury and the resultant extent of myocardial damage. Our findings suggest that high plasma BNP levels might have a clinically important protective effect on ischemic myocardium in patients with STEMI who receive percutaneous coronary intervention., (© The European Society of Cardiology 2015.)

The incidence of chronic lymphocytic leukemia (CLL) is low in Japan. The clinical course ranges from very indolent to rapidly progressive. Recently, several reports have indicated that mutation of the splicing factor 3b, subunit 1 (SF3B1) gene in CLL is predictive of a poor prognosis. Here, we investigated the SF3B1 mutational status of Japanese CLL patients and clarified the association between SF3B1 mutational status and prognostic factors. One hundred and two patients that were referred to our institutions between 1999 and 2013 were enrolled. Mutation analysis of SF3B1 (n = 87) and of the immunoglobulin heavy chain gene (IGHV) (n = 102) was performed at diagnosis. FISH analysis of del(11)(q22) was performed for 17 patients. Seven patients have SF3B1 mutation (8.0 %: K700E, 5/7; G742D, 1/7 and Y623C, 1/7). The median survival times for patients with mutated and non-mutated SF3B1 were 53 and 130 months, respectively. Overall survival of the mutated SF3B1 group was significantly lower than that of the non-mutated group (p = 0.0187). No relationship was observed between IGHV mutational status and SF3B1 mutation. There was no patient with SF3B1 mutation in the IGHV1-69 population (0/2). In conclusion, mutation of SF3B1 at diagnosis in Japanese CLL patients is predictive of a poor prognosis.

Chemical composition and potent odorants that contribute to the characteristic odor of essential oil from Alismatis Rhizoma (tubers of Alisma orientale) were investigated by gas chromatography-mass spectrometry (GC-MS), GC-olfactometry (GC-O), aroma extract dilution analysis (AEDA) and relative flavor activity (RFA) methods. Fifty components, representing 94.5% of the total oil, were identified. In this study, we newly identified thirty-nine compounds in the oil from tubers of A. orientale. The major constituents of the essential oil were khusinol (36.2%), δ-elemene (12.4%), germacron (4.1%), alismol (3.8%), β-elemene (3.1%), and α-bisabolol (1.9%). Through sensory analysis, sixteen aroma-active compounds were detected and the key contributing aroma-active compounds were δ-elemene (woody, flavor dilution (FD)-factor = 4, RFA = 0.3) β-elemene (spicy, FD = 5, RFA = 0.7), spathulenol (green, FD = 5, RFA = 1.0), γ-eudesmol (woody, FD = 6, RFA = 1.5), and γ-cadinol (woody, FD = 5, RFA = 1.0). These compounds are thought to contribute to the odor from tubers of A. orientale. These results imply that the essential oil from the tubers of A. orientale deserve further investigations in the phytochemical and medicinal fields.

A 45-year-old man was admitted to our hospital because of taste disorders in March 2014. He exhibited cervical muscle weakness and left eye ptosis, which responded to Tensilon test, and was diagnosed with myasthenia gravis (MG). He developed aspiration pneumonia and myasthenic crisis, which was treated with intravenous immunoglobulin and steroid pulse therapy. All symptoms disappeared. Oral administration of prednisolone and tacrolimus was started. Chest CT revealed thymoma and extended thymectomy was performed in May 2014. In December 2014, seven months after the thymectomy, hematological examination showed pancytopenia including severe neutropenia. We diagnosed his illness as aplastic anemia (AA). Cyclosporine therapy with transfusion was administerd and led to reticulocyte count recovery. Since May 2015, hemoglobin recovery reached a blood transfusion free period. To our knowledge, this is the first case report with the patient supposed of relationship among taste disorders, AA and thymoma-associated MG.

The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes., (Copyright © 2015 John Wiley & Sons, Ltd.)

The aim of this study was to investigate the chemical composition and the odor-active components of volatile oils from three edible mushrooms, Pleurotus ostreatus, Pleurotus eryngii, and Pleurotus abalonus, which are well-known edible mushrooms. The volatile components in these oils were extracted by hydrodistillation and identified by GC/MS, GC-olfactometry (GC-O), and aroma extract dilution analysis (AEDA). The oils contained 40, 20, and 53 components, representing 83.4, 86.0, and 90.8% of the total oils in P. ostreatus, P. eryngii, and P. abalonus, respectively. Odor evaluation of the volatile oils from the three edible mushrooms was also carried out using GC-O, AEDA, and odor activity values, by which 13, eight, and ten aroma-active components were identified in P. ostreatus, P. eryngii, and P. abalonus, respectively. The most aroma-active compounds were C8 -aliphatic compounds (oct-1-en-3-ol, octan-3-one, and octanal) and/or C9 -aliphatic aldehydes (nonanal and (2E)-non-2-enal)., (Copyright © 2015 Verlag Helvetica Chimica Acta AG, Zürich.)

A novel methodology for the sensing of formaldehyde that displays a response using distinct and diverse color changes is reported. Through copolymerization of a primary amine monomer with additional co-monomers on a pattern-printed microscope slide, primary amine-containing thin films were obtained. After the absorption of a range of colors of anionic dyes, the thin films were immersed in aqueous formaldehyde solutions. It was demonstrated that the color of the thin films changed depending on the formaldehyde concentration in the solution. As the anionic dyes were released from the thin films at varying formaldehyde concentrations, a set of thin films exhibiting a range of color-change patterns was observed. The response selectivity of the thin films towards carbonyl compounds was examined, and sensitivity in the order of formaldehyde»acetaldehyde>acetone was observed. In addition, the effect of amine structure was examined, and it was found that thin films bearing tertiary amino groups show virtually no formaldehyde response. These observations clearly indicate that the existence of primary amino groups is essential for color changes to be observed, and that the formation of an imine is the crucial step in generating a response against formaldehyde. The formaldehyde-responsive system presented herein is advantageous, as its preparation is relatively simple and does not require complex organic synthesis. In addition, a wide range of anionic dyes is compatible with the system, and can be selected in terms of color, charge, toxicology profile, and cost, for example., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: In animal models of acute myocardial infarction, n-3 polyunsaturated fatty acids (PUFAs) administered before coronary occlusion have been suggested to prevent induction of ventricular arrhythmia and limit infarct size. However, the relation between the serum levels of n-3 PUFAs and ischemia/reperfusion (I/R) injury remains unclear., Methods: 211 patients with ST-segment elevation acute myocardial infarction received emergency percutaneous coronary intervention (PCI) within 6h from the onset. The patients were divided into two groups according to the sum of serum eicosapentaenoic acid (EPA) levels and docosahexaenoic acid (DHA) levels before PCI: group L (n=106), EPA+DHA <155μg/ml and group H (n=105), EPA+DHA ≥155μg/ml. The Selvester QRS-scoring system was used to estimate the serial change in infarct size., Results: Time to reperfusion was similar between the 2 groups. The QRS score before PCI was higher in group L than in group H (2.42±2.00 vs 1.85±2.01, p=0.015). The proportion of patients with I/R injury immediately after reperfusion, defined as reperfusion ventricular arrhythmias (25% vs 11%, p=0.006) and ST-segment re-elevation (44% vs 22%, p<0.001), was also higher in group L than in group H, followed by a greater increment in the QRS score during PCI (3.51±2.51 vs 2.54±1.91, p=0.006) and higher peak levels of creatinine phosphokinase (3552±241U/L vs 2660±242U/L, p<0.01). On multivariate analysis, serum level of EPA+DHA was an independent predictor of reperfusion injury (odds ratio 0.985, p=0.032)., Conclusion: Serum level of n-3 PUFAs before PCI may be a predictor of I/R injury and the resultant extent of myocardial damage. These findings suggest a protective effect of serum n-3 PUFAs on ischemic myocardium., (Copyright © 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Two sample preparation methods, namely hydrodistillation (HD) and solvent-assisted flavor evaporation (SAFE), have been used to investigate the essential oils of the aerial parts (leaves and stems) of Symplocarpus foetidus, a plant with a characteristic odor, by gas chromatography mass spectrometry (GC-MS). Characteristic aroma-active compounds in the oils were detected by GC-Olfactometry (GC-O) and aroma extract dilution analysis (AEDA). From the HD method, the main compounds in the oil were found to be p-vinyl-guaiacol (15.5%), 2-pentyl-furan (13.4%), and (Z)-ligustilide (9.5%). From the SAFE method, the main compounds were 2-butoxy-ethanol (49.6%), ethyl-pentanoate (4.5%), and mesitylene (4.0%). In HD oil, the most intense aroma-active compounds were 2-pentyl-furan (flavor dilution factor (FD) = 32, odor activity value (OAV) = 57), p-vinyl-guaiacol (FD = 16, OAV = 41), and dimethyl disulfide (FD = 16, OAV = 41). In SAFE oil, the main aroma-active compounds were 2-butoxy ethanol (FD = 32, OAV = 16), and 2-methoxy thiazole (FD = 32, OAV = 25).

This study was investigated the chemical composition of volatile oils and aroma evaluation from the tubers of Apios americana Medikus. Theses volatile oils were obtained by the hydrodistillation (HD) and the solvent-assisted flavor evaporation (SAFE) methods. These oils were analyzed by Gas chromatography (GC), GC-mass spectrometry (GC-MS), GC-olfactometry (GC-O), aroma extract dilution analysis (AEDA) and odor activity values (OAV) for the first time. The major compounds in the HD oil were palmitic acid (36.5%), linoleic acid (10.5%) and nonadecanol (5.7%). Meanwhile, in the SAFE oil, the major compounds were 4-hydroxy-4-methyl-2-pentanone (34.2%), hexanal (11.0%) and hexanol (7.9%). Through aroma evaluation, 20 (HD) and 14 (SAFE) aroma-active compounds were identified by GC-O. As a result, the most intense aroma-active compounds in both extraction methods were 1-octen-3-ol and hexanal, both of which showed high odor activity values (OAV).

The present study focuses on the volatile compounds with characteristic odor of essential oil from the leaves of Magnolia obovata by hydrodistillation (HD) and solvent-assisted flavor evaporation (SAFE) method. Eighty-seven compounds, representing 98.0% of the total oil, were identified using HD. The major compounds of HD oil were (E)-β-caryophyllene (23.7%), α-humulene (11.6%), geraniol (9.1%), and borneol (7.0%). In SAFE oil, fifty-eight compounds, representing 99.7% of the total oil, were identified. The main compounds of SAFE oil were (E)-β-caryophyllene (48.9%), α-humulene (15.7%), and bicyclogermacrene (4.2%). In this study, we newly identified eighty-five compounds of the oils from M. obovata leaves. These oils were also subjected to aroma evaluation by gas chromatography-olfactometry (GC-O) and aroma extract dilution analysis (AEDA). As a result, twenty-four (HD) and twenty-five (SAFE) aroma-active compounds were detected. (E)-β-Caryophyllene, α-humulene, linalool, geraniol, 1,8-cineole, and bicyclogermacrene were found to impart the characteristic odor of M. obovata leaves. These results imply that the oils of M. obovata leaves must be investigated further to clarify their potential application in the food and pharmaceutical industries.

The chemical compositions of essential oils from the flower and aerial parts (i.e., leaf and branch) of Eurya japonica were determined and quantified using gas chromatography-mass spectrometry (GC-MS). A total of 87 and 50 compounds were detected in the oils from the flower and aerial parts, respectively. The main compounds of the flower oil were linalool (14.0%), (9Z)-tricosene (12.0%), and nonanal (7.4%). In the oil from the aerial parts, linalool (37.7%), α-terpineol (13.5%), and geraniol (9.6%) were detected. In the oils from the flower and aerial parts, 13 and 8 aroma-active compounds were identified by GC-olfactometry (GC-O) analysis, respectively. The key aroma-active compounds of the flower oil were heptanal [fatty, green, flavor dilution (FD) = 128, odor activity value (OAV) = 346], nonanal (sweet, citrus, FD = 128, OAV = 491), and eugenol (sweet, spicy, FD = 64, OAV = 62): in the oil from the aerial parts, the key aroma-active compounds were linalool (sweet, citrus, FD = 64, OAV = 95), (E)-β-damascenone (sweet, FD = 256, OAV = 4000), and (E)-β-ionone (floral, violet, FD = 128, OAV = 120). This study revealed that nonanal and eugenol impart the sweet, citrus, and spicy odor of the flower oil, while (E)-β-damascenone and (E)-β-ionone contribute the floral and sweet odor of the oil from the aerial parts.

Introduction: The Chrysanthemum genus consisting of about 200 species is mainly distributed over the Northern Hemisphere. Despite the pleasant odour of C. japonense var. debile (setonojigiku), no detailed analysis of the aroma-active compounds has been reported using sensory evaluation., Objectives: Using a hydrodistillation (HD) and a solvent-assisted flavour evaporation (SAFE) method to obtain the volatile oil from the leaf parts., Methods: To clarify odorants contributing to the characteristic aroma-active compounds, the aroma-extract dilution analysis (AEDA) method was performed through gas chromatography olfactometry (GC/O) analysis. In addition, the odour activity value (OAV) was calculated in order to determine the relative contribution of each compound to the aroma-active compounds., Results: A total of 42 components by HD oil were identified by GC-MS, whereas 34 components were identified in SAFE oil. Thirteen compounds were identified by GC/O analysis in HD and SAFE oils respectively., Conclusion: Each extraction method has its own advantages and disadvantages, and they are generally complementary to each other. On the basis of AEDA, OAV and sensory evaluations, [2.2.1] bicyclic monoterpenes (borneol, bornyl acetate and camphor) and β-caryophyllene are considered to be the main aroma-active compounds of both extraction methods., (Copyright © 2014 John Wiley & Sons, Ltd.)

In this study, the characteristic odorants of the volatile oils from Pleurotus species (P. eryngii var. tuoliensis and P. cystidiosus) were extracted by hydrodistillation and analyzed by gas chromatography-mass spectrometry (GC-MS), gas chromatography-olfactometry (GC-O), and aroma extract dilution analysis (AEDA). A total of 52 and 54 components (P. eryngii var. tuoliensis and P. cystidiosus, respectively) were identified, representing about 98.8% and 85.1% of the volatile oils, respectively. The main components of the P. eryngii var. tuoliensis oil were palmitic acid (82, 38.0%), oleic acid (86, 25.0%) and linoleic acid (85, 9.7%). The main components of the P. cystidiosus oil, palmitic acid (82, 25.8%), indole (54, 9.1%) and myristic acid (77, 5.3%). Regarding the aroma components, 16 and 13 components were identified in the P. eryngii var. tuoliensis and P. cystidiosus oils respectively, by the GC-O analyses. The results of the sniffing test, odor activity value (OAV) and flavor dilution (FD) factor indicate that methional, 1-octen-3-ol and nonanal are the main aroma-active components of P. eryngii var. tuoliensis oil. On the other hands, dimethyl trisulfide and 1-octen-3-ol were estimated as the main aroma-active components of the P. cystidiosus oil.

The chemical composition of the volatile oil extracted from the aerial parts of Brassica rapa cv. "yukina" was analyzed using GC-MS, GC-PFPD, and GC-O. A total of 50 compounds were identified. The most prominent constituents were (E)-1,5-heptadiene (40.27%), 3-methyl-3-butenenitrile (25.97%) and 3-phenylpropanenitrile (12.41%). With regard to aroma compounds, 12 compounds were identified by GC-O analysis. The main aroma-active compounds were dimethyl tetrasulfide (sulphury-cabbage, FD = 64), 3-phenylpropanenitrile (nutty, FD = 64), 3-methylindole (pungent, FD = 64), and methional (potato, FD = 32). The antioxidant activity of the aroma-active compounds of the oil was determined using an oxygen radical absorbance capacity (ORAC) assay using fluorescein as the fluorescent probe. The ORAC values were found to be 785 ± 67 trolox equivalents (μmol TE/g) for B. rapa cv. "yukina" oil. The results obtained showed that the volatile oil extracted from the aerial parts is a good dietary source of antioxidants.

A 41-year-old man was admitted with decompensated heart failure. Mechanical ventilation was maintained with a large dose of propofol. On day 4, significant ST elevation with complete atrioventricular block was noted, which subsequently induced cardiopulmonary arrest. Treatment with percutaneous cardiopulmonary support and therapeutic hypothermia was initiated. Emergent cardiac angiography showed simultaneous multivessel coronary spasms. Although nitroglycerin and nicorandil were ineffective, the intracoronary administration of fasudil, a Rho-kinase inhibitor, successfully resolved the vasospasms. However, during rewarming, the coronary vasospasms recurred, and the patient died of cardiogenic shock. In addition to hypertrophy, the autopsied heart demonstrated the accumulation of inflammatory cells in the pericardium and adventitia of the coronary arteries.

This study is focused on the volatile oils from the fruiting bodies of Pleurotus salmoneostramineus (PS) and P. sajor-caju (PSC), which was extracted by hydrodistillation (HD) and solvent-assisted flavor evaporation (SAFE) methods. The oils are analyzed by gas chromatography-mass spectrometry (GC-MS), GC-olfactometry (GC-O), and aroma extract dilution analysis (AEDA). A total of 31, 31, 45, and 15 components were identified in PS (HD and SAFE) and PSC (HD and SAFE), representing about 80.3%, 92.2%, 88.9%, and 83.0% of the oils, respectively. Regarding the aroma-active components, 13, 12, 13, and 5 components were identified in PS (HD and SAFE) and PSC (HD and SAFE), respectively, by the GC-O analyses. The results of the sniffing test, odor activity value (OAV) and flavor dilution (FD) factor indicate that 1-octen-3-ol and 3-octanone are the main aroma-active components of PS oils. On the other hands, methional and 1-octen-3-ol were estimated as the main aroma-active components of PSC oils.

The chemical composition of volatile oil from agitake (Pleurotus eryngii var. ferulae) was established for the first time using gas chromatography (GC) and GC-mass spectrometry. Sixty-seven and 24 components were extracted by hydrodistillation (HD) using diethyl ether (DE) and dichloromethane (DM), respectively; these components accounted for 80.3% and 91.8% of the total oil, respectively. Thirteen and 48 components of were extracted by the solvent-assisted flavor evaporation method (SAFE), using DE and DM, respectively, and identified; these components accounted for 83.5% and 82.0% of the total oil, respectively. Methylsuccinimide and 2,3,7-trimethyl-2-octene were the most characteristic components by SAFE using DM.Odor evaluation of the volatile oil from agitake was also carried out using GC-olfactometry (GC-O), aroma extraction dilution analysis (AEDA), and the odor activity value (OAV). Sixteen, 8, 5 and 9 aroma-active components were identified using HD (DE and DM) and SAFE (DE and DM), respectively. The main aroma-active components extracted using HD and SAFE were 1-octen-3-ol (mushroom-like) and phenylacetaldehyde (floral), respectively. This study proved that HD and SAFE can be used as complementary extraction techniques for the complete characterization of volatile oil from agitake.

The in vitro biotransformation of karahanaenone was examined in cytochrome P450 (CYP) 2A6. The biotransformation of karahanaenone by CYP2A6 was investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). Karahanaenone was found to be oxidized to two metabolites by CYP2A6. In order to produce large quantity of metabolites by CYP2A6, the biotransformation of karahanaenone by Salmonella typhimurium OY1002/2A6 was investigated. Similarly, two metabolites were confirmed by GC and GC-MS. The structure of metabolites was determined by 1D NMR, 2D NMR, and infrared, as a result there were new compounds, (6R)-hydroxykarahanaenone and 10-hydroxykarahanaenone.

The components of the essential oil from the roots of Alangium salviifolium were analyzed by capillary gas chromatography-mass spectrometry (GC-MS). Ninety compounds, representing 74.5% of the total oil, were identified; the main components of the oil were epi-α-cadinol, followed by trans-2-hydroxycalamenene, cadalene, and cadina-4,10(15)-dien-3-one. A further unknown component comprised 5.5% of the oil. Therefore, the essential oil was purified by flash column chromatography to isolate this component. Its structure was established using extensive spectroscopic data analyses, including NMR, HR-EI-MS, and IR. The results showed that this isolated compound was (-)-7, 8-dihydroxycalamenal, which is a novel cadinane-type sesquiterpenoid. This compound was tested for its antioxidant activity and inhibition of tyrosinase, and showed particularly strong inhibition effects.

A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny. However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular functions in a tissue-specific manner., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The "CTCF code" hypothesis posits that CTCF pleiotropic functions are driven by recognition of diverse sequences through combinatorial use of its 11 zinc fingers (ZFs). This model, however, is supported by in vitro binding studies of a limited number of sequences. To study CTCF multivalency in vivo, we define ZF binding requirements at ∼50,000 genomic sites in primary lymphocytes. We find that CTCF reads sequence diversity through ZF clustering. ZFs 4-7 anchor CTCF to ∼80% of targets containing the core motif. Nonconserved flanking sequences are recognized by ZFs 1-2 and ZFs 8-11 clusters, which also stabilize CTCF broadly. Alternatively, ZFs 9-11 associate with a second phylogenetically conserved upstream motif at ∼15% of its sites. Individually, ZFs increase overall binding and chromatin residence time. Unexpectedly, we also uncovered a conserved downstream DNA motif that destabilizes CTCF occupancy. Thus, CTCF associates with a wide array of DNA modules via combinatorial clustering of its 11 ZFs., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Lymphocyte activation is initiated by a global increase in messenger RNA synthesis. However, the mechanisms driving transcriptome amplification during the immune response are unknown. By monitoring single-stranded DNA genome wide, we show that the genome of naive cells is poised for rapid activation. In G0, ∼90% of promoters from genes to be expressed in cycling lymphocytes are polymerase loaded but unmelted and support only basal transcription. Furthermore, the transition from abortive to productive elongation is kinetically limiting, causing polymerases to accumulate nearer to transcription start sites. Resting lymphocytes also limit the expression of the transcription factor IIH complex, including XPB and XPD helicases involved in promoter melting and open complex extension. To date, two rate-limiting steps have been shown to control global gene expression in eukaryotes: preinitiation complex assembly and polymerase pausing. Our studies identify promoter melting as a third key regulatory step and propose that this mechanism ensures a prompt lymphocyte response to invading pathogens., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Activation-induced cytidine deaminase (AID) promotes chromosomal translocations by inducing DNA double-strand breaks (DSBs) at immunoglobulin (Ig) genes and oncogenes in the G1 phase. RPA is a single-stranded DNA (ssDNA)-binding protein that associates with resected DSBs in the S phase and facilitates the assembly of factors involved in homologous repair (HR), such as Rad51. Notably, RPA deposition also marks sites of AID-mediated damage, but its role in Ig gene recombination remains unclear. Here, we demonstrate that RPA associates asymmetrically with resected ssDNA in response to lesions created by AID, recombination-activating genes (RAG), or other nucleases. Small amounts of RPA are deposited at AID targets in G1 in an ATM-dependent manner. In contrast, recruitment in the S-G2/M phase is extensive, ATM independent, and associated with Rad51 accumulation. In the S-G2/M phase, RPA increases in nonhomologous-end-joining-deficient lymphocytes, where there is more extensive DNA-end resection. Thus, most RPA recruitment during class switch recombination represents salvage of unrepaired breaks by homology-based pathways during the S-G2/M phase of the cell cycle., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

We evaluated the prognostic significance of the serum level of the soluble form of interleukin-2 receptorα (sIL-2Rα) and investigated its association with CD25 expression on tumor cells in diffuse large B-cell lymphoma (DLBCL). Three hundred and thirty-eight adult patients with newly diagnosed DLBCL were eligible for this retrospective study. 32.2% of patients were treated with CHOP-like regimen and 67.8% with R-CHOP-like regimen. CD25 expression on the surface of tumor cells was evaluated in 143 cases and its relationship with sIL-2Rα level was also investigated. Both overall survival (OS) and progression-free survival (PFS) were poorer in patients with higher sIL-2Rα, in both R-CHOP and CHOP groups. sIL-2Rα > 1,000 U/mL and performance status (PS) ≥ 2 were independently associated with poorer OS, and sIL-2Rα > 1,000 U/mL, age > 60 years, and ≥ 2 extranodal sites were independently associated with poorer PFS in the R-CHOP group. The sIL-2Rα level was higher in the CD25-positive group than in the CD25-negative group in stage 3 or 4 disease (p = 0.010). Multiple linear regression analysis showed CD25 expression to be independently correlated with sIL-2Rα levels. High sIL-2Rα is an important risk factor for survival in DLBCL treated with not only CHOP-like, but also R-CHOP-like regimens, regardless of the tumor's expression of CD25.

In this study, biotransformation of (+)-fenchone (compound 1) by Salmonella typhimurium OY1002/2A6 expressing human CYP2A6 and NADPH-P450 reductase yielded two oxidized metabolites, namely, (+)-(1S,6R)-6-endo-hydroxyfenchone (compound 2) and (+)-(1S,6S)-6-exo-hydroxyfenchone (compound 3). The conversion rate of compound 1 to compound 2 and 3 was 2.4% and 5.2%, respectively. This is the first study that succeeded in metabolizing compound 1 to obtain large amounts of metabolite 2 and 3 by using S. typhimurium OY1002/2A6 expressing human CYP2A6 and NADPH-P450 reductase.

The components of the essential oil from the roots of Pueraria mirifica were analyzed by capillary gas chromatography-mass spectrometry (GC-MS). Eighty-two components, representing 88.5% of the total oil, were identified by GC-MS. The main component of the oil was 2-pentylfuran, followed by hexanal and hexadecanol. With regard to the odor components from the essential oil of P. mirifica as determined by gas chromatography-olfactometry and aroma extract dilution analysis, it was revealed that phenylacetaldehyde and (2E)-nonenal imparted the green odor of the oil, and geraniol contributed to the sweet odor.

Objectives: Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by the production of autoreactive antibodies against platelet antigens. Although dysfunction of multiple aspects of cellular immunity is considered to be important in the pathogenesis of ITP, it has not been clarified which cell types play a principal role., Methods: We enrolled 46 untreated patients with chronic ITP and 47 healthy adult volunteers, and investigated by flow cytometry the percentage and absolute number of cells in their peripheral blood that participate in the regulation of cellular immunity. These included plasmacytoid dendritic cells (pDCs), myeloid dendritic cells (mDCs), natural killer (NK) cells, natural killer T (NKT) cells, regulatory T (Treg) cells, and Th17 cells., Results: We found a significant reduction in the absolute number of pDCs, but not of mDCs, in patients with ITP when compared with healthy controls (P < 0.001). Reduced numbers of circulating pDCs were observed in both Helicobacter pylori (H. pylori)-positive and Helicobacter pylori (H. pylori)-negative patients with ITP. In contrast, there were no significant differences in the numbers of circulating Treg cells, Th17 cells, NK cells, or NKT cells. Interestingly, we observed increases in the number of pDCs after H. pylori eradication by antibiotics in responders but not in non-responders, while pDCs and mDCs decreased markedly after prednisolone therapy in both responders and non-responders. In patients without treatment, low pDC numbers persisted during the observational period., Conclusions: We demonstrated that the number of circulating pDCs is low in patients with primary and H. pylori-associated ITP and that it changes depending on treatment modality. Further investigation is warranted with regard to the role of pDCs in the immunopathogenesis of ITP., (© 2011 John Wiley & Sons A/S.)

We retrospectively investigated pathological types, clinical backgrounds, treatments and prognoses in 726 adult patients with newly diagnosed malignant lymphoma in Gunma Prefecture. They consisted of 679 patients with non-Hodgkin lymphoma (B-cell type, 603; T- and NK-cell type, 76) of which 376 patients had diffuse large B-cell lymphoma (DLBCL) and 47 patients with Hodgkin lymphoma. When comparing the prognosis of DLBCL between patients receiving rituximab (R-CHOP group; n=212) and not using rituximab (CHOP group; n=126), both 3-year overall survival (73.5% vs 61.7%, p=0.010) and 3-year progression-free survival (65.1% vs 45.8%, p<0.001) were statistically better in the R-CHOP group compared to the CHOP group. Our results suggest that more than half of patients were DLBCL and the rituximab-containing regimen results in an improved prognosis for DLBCL patients.

Recurrent chromosomal translocations underlie both haematopoietic and solid tumours. Their origin has been ascribed to selection of random rearrangements, targeted DNA damage, or frequent nuclear interactions between translocation partners; however, the relative contribution of each of these elements has not been measured directly or on a large scale. Here we examine the role of nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured mouse B lymphocytes. In the absence of recurrent DNA damage, translocations between Igh or Myc and all other genes are directly related to their contact frequency. Conversely, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA break formation, as measured by replication protein A accumulation at the site of damage. Thus, non-targeted rearrangements reflect nuclear organization whereas DNA break formation governs the location and frequency of recurrent translocations, including those driving B-cell malignancies.

T-cell prolymphocytic leukemia (T-PLL) is a rare type of peripheral T-cell leukemia. In this study, we examined the clinical and biological characteristics of 11 Japanese patients with T-PLL. Median age was 74 years, with male predominance. Median lymphocyte frequency was 85.3% in blood. Physical characteristics were splenomegaly (36.4%), tiny lymph adenopathy (63.6%), skin lesion (9.1%) and pleural effusion (27.3%). Median survival was 30.1 months, despite treatment with various chemotherapeutic modalities. Although complex chromosomal abnormalities were observed in 5 of 11 cases (45.5%), typical 14q32 and Xq28 abnormalities were not detected. TCL1A mRNA expression was observed in 6 of 11 cases (54.5%) on real-time quantitative PCR. In 5 of these 6 cases, flow cytometric analysis and/or immunohistochemistry confirmed the expression of TCLA1 protein. Split signals for the TCL1 region on fluorescence in situ hybridization confirmed rearrangement in 3 out of 7 cases evaluated. These cases corresponded to cases that were positive for TCL1A expression, suggesting that rearrangement of the TCL1 region induced high expression of TCL1A gene. In summary, a substantial number of T-PLL cases in Japan had abnormal expression of TCL1A, probably due to rearrangement of TCL1 region. Expression and/or rearrangement of TCL1A may, therefore, be a useful marker for diagnosing T-PLL, regardless of chromosomal abnormalities.

Chromosomal rearrangements, including translocations, require formation and joining of DNA double strand breaks (DSBs). These events disrupt the integrity of the genome and are frequently involved in producing leukemias, lymphomas and sarcomas. Despite the importance of these events, current understanding of their genesis is limited. To examine the origins of chromosomal rearrangements we developed Translocation Capture Sequencing (TC-Seq), a method to document chromosomal rearrangements genome-wide, in primary cells. We examined over 180,000 rearrangements obtained from 400 million B lymphocytes, revealing that proximity between DSBs, transcriptional activity and chromosome territories are key determinants of genome rearrangement. Specifically, rearrangements tend to occur in cis and to transcribed genes. Finally, we find that activation-induced cytidine deaminase (AID) induces the rearrangement of many genes found as translocation partners in mature B cell lymphoma., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The in vitro metabolism of (+)-(1S,3S,4R) and (-)-(1R,3R,4S)-menthol enantiomers was examined by incubation with human liver microsomes, and the oxidative metabolites thus formed were analyzed using gas chromatography-mass spectrometry (GC-MS). The (+)- and (-)-menthols were found to be oxidized to the respective (+)-(1S,3S,4S)- and (-)-(1R,3R,4R)-trans-p-menthane-3,8-diol derivatives by human liver microsomal P450 enzymes. Cytochrome P450 (CYP) 2A6 was determined to be the major enzyme involved in the hydroxylation of (+)- and (-)-menthols by human liver microsomes on the basis of the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 catalyzed the oxidation of (+)- and (-)-menthols. Second, oxidation of (+)- and (-)-menthols was inhibited by (+)-menthofuran and anti-CYP2A6 antibody. Finally, (+)- and (-)-menthol activities were found to correlate with contents of CYP2A6 in liver microsomes of 9 human samples.

Vincristine-adriamycin-dexamethasone (VAD) regimen with intermittent high-dose dexamethasone (HD) has been used as primary chemotherapy for multiple myeloma (MM) patients who are candidates for high-dose therapy or present with renal failure. However, dexamethasone increases the risk of infection in MM patients. We retrospectively evaluated treatment efficacy and infectious events in MM patients undergoing VAD with or without HD. Seventy-seven consecutive patients who received VAD without HD (n = 37) or VAD-HD (n = 40) at our institution were assessed. Characteristics of patients and VAD regimens were retrospectively analyzed to detect correlations with the incidence of infections. During 218 VAD cycles, 48 infectious episodes were documented in 39 patients. Of these, 32 episodes in 26 patients were severe (grade ≥ 3). By analyzing each patient, VAD-HD was associated with risk of all-grade and severe bacterial infection, while International Staging System stage ≥ 2 was independently correlated with severe bacterial infection. Response rates after two cycles were comparable between the 2 VAD regimens. In conclusion, risk of infection is lower in VAD without HD than in VAD-HD, and the clinical response is equivalent. VAD-HD should thus be avoided for MM patients with high risk of infection.

In a previous in vitro study, (-)-camphor (1) was examined by incubation with human liver microsomes, and the oxidative metabolites thus formed were analyzed using gas chromatography-mass spectrometry. However, thus far, no large-scale biotransformation using recombinant human P450 has been performed. Here, the biotransformation of compound 1 has been investigated by using Salmonella typhimurium OY1002/2A6 expressing human CYP2A6 and human NADPH-P450 reductase as a biocatalyst. Compound 1 (400 mg) was converted to (1S,5S)-(-)-5-exo-hydroxycamphor (2) (30.4 mg) and (1S,7S)-(-)-8-hydroxycamphor (3) (2.4 mg) by S. typhimurium OY1002/2A6. This is the first report to show that large quantities of metabolites 2 and 3 can be produced by S. typhimurium OY1002/2A6 expressing human CYP2A6 and NADPH-P450 reductase.

Telomerase activity has been found in most common cancers, thus indicating that telomerase detection may be a useful marker in cancer diagnosis. The telomeric amplification protocol (TRAP) assay and RT-PCR are customarily used to detect telomerase activity and the expression of the associated genes in cells. However, these methods do not provide any information about telomerase activation at an individual cell level. To analyze cells separately, those cells have to be isolated by sometimes complicated method. The immunohistochemical detection of human telomerase reverse transcriptase (hTERT) is useful to detect telomerase positive cells in a background of non-cancerous cells. A method has been developed for the detection of intranuclear hTERT protein, in a subpopulation of hematopoietic cells, using concurrent staining of a cell surface antigen and multicolor flow cytometry. Only mouse monoclonal anti-hTERT antibody demonstrated the specific positivity in immunocytochemistry and immunofluorescent flow cytometry. Human leukemia and myeloma cell lines showed 100% positivity, whereas normal neutrophils showed 0% positivity. hTERT expression was analyzed in hematopoietic precursor cells of bone marrow samples using concurrent staining of surface CD34 antigen and intracellular hTERT protein and multi-parameter flow cytometry. CD34 positive cells demonstrated higher expression of hTERT than CD34 negative cells. A quick, easy and sensitive assay for determining the hTERT protein expression has been developed. Using this method and the multi-parameter nature of flow cytometry and its ability to identify cellular subpopulations will provide a better understanding of the mechanisms regarding the activation of telomerase., (Copyright 2009 Elsevier Ltd. All rights reserved.)

In Japan, typical hairy cell leukemia (HCL) is rare, and HCL-Japanese variant (HCL-JV) is more common. Hairy B-cell lymphoproliferative disorder (HBLD) is another unusual disorder of polyclonal B-lymphocytosis of hairy cell appearance. In the present study, we analyzed the clinical features of 3 patients with HCL, 3 with HCL-JV, and 3 with HBLD. All HBLD patients had the DRB1*04 allele. As compared with other B-cell lymphoproliferative disorders, CD27 expression on B cells was significantly lower in all patients, ranging from 0.3% to 23.4%. Our results suggest that low CD27 expression may be a distinct feature of these HCL-related disorders.

The incidence of chronic lymphocytic leukemia is low in the Japanese population compared with populations in western countries, suggesting a role for genetic factors in the occurrence of this disease. We have previously shown that chronic lymphocytic leukemia in Japan rarely expresses the immunoglobulin heavy chain variable region (IGHV) 1-69 gene (1 out of 43 patients, 2.3%), which is a gene most commonly expressed in chronic lymphocytic leukemia cases from western countries. In the current study, we extended the previous study by examining immunoglobulin heavy chain and light chain gene expression in 80 Japanese patients with chronic lymphocytic leukemia and in 52 Japanese patients with other leukemic chronic lymphoproliferative disorders. IGHV1-69 gene expression was again quite low in our cohort, found in only two patients: one with chronic lymphocytic leukemia and the other with splenic marginal zone lymphoma. The IGHV4-34 gene was most frequently expressed in chronic lymphocytic leukemia (27.5%), whereas it was rarely found in leukemic chronic lymphoproliferative disorders (7.7%, P = 0.005). There was also a significant difference in the expression of IGLV3-21 between chronic lymphocytic leukemia and leukemic chronic lymphoproliferative disorders (29.4 vs 4.8%, P = 0.018). The IGLV3-21 gene in the majority of chronic lymphocytic leukemia cases was associated with homologous complementarity determining region 3 sequences. Recent studies identified subsets of cases expressing almost identical B-cell receptors. We found that two patients with chronic lymphocytic leukemia and the patient with splenic marginal zone lymphoma expressed IGHV4-39/IGKV1-39 and IGHV1-69/IGKV3-20, respectively, which belong to these subsets.

A 72-year-old woman was referred to our hospital for evaluation of leukocytosis revealed by a medical examination. Her physical examination demonstrated no splenomegaly and no palpable lymph nodes. Her white cell count was 10,900/microl with atypical lymphocytosis (84.5%). Her hemoglobin concentration was 10.4 g/dl, and platelet count 151,000/microl. On peripheral blood smears, the atypical lymphocytes had a hairy cell-like appearance, and phase-contrast microscopic and transmission electron microscopic findings revealed the lymphocytes had many long surface microvilli. Flowcytometric analysis of peripheral blood lymphocytes identified expanded B-lymphocytes as having the IgG+, CD5- CD10- CD11c+ CD19+ CD20+ CD23- CD25- and CD103- cell surface phenotype. Serum electrophoresis disclosed polyclonal elevation of IgG and IgM (2620 mg/dl and 840 mg/dl, respectively). No light-chain restriction and a polyclonal VH gene rearrangement pattern indicated the polyclonal proliferation of B cells. The patient was a nonsmoker and had HLA-DR4, as in previous reports which have suggested an association between hairy B-cell lymphoproliferative disorder (HBLD) and HLA-DR4. No chromosome 3 abnormality was observed. These findings were consistent with the characteristics of HBLD, but differed in some respects from those of persistent polyclonal B-cell lymphocytosis (PPBL). Therefore, we diagnosed this patient as having HBLD.

Hospice ward at Matsuyama Bethel Hospital opened in April 2000. The hospice care has been provided for inpatients and outpatients. We considered a system that should be established to allow patients and their families to choose from in-hospital care, outpatient care and home care. The hospice consultation for outpatients opened in April 2004. The terminal cancer patients who are within 6 months of remaining days were hospitalized to the hospice ward. After making the hospice consultation for outpatients, hospice care services have been provided for terminal cancer patients including those with more than 6 months of remaining days under the coordination of palliative care for outpatients and inpatients.

Surgically resected specimens of 13 carcinoid tumors of the lung including nine typical carcinoids and four atypical carcinoids, and eight salivary gland type carcinomas (six mucoepidermoid carcinomas and two adenoid cystic carcinomas) were analyzed regarding p53 expression, loss of heterozygosity (LOH) in chromosome 3p, 9p, and K-ras mutation. The overexpression of p53 was identified in four atypical carcinoid tumors, one mucoepidermoid carcinoma, and one adenoid cystic carcinoma, however, none of typical carcinoids showed p53 immunoreactivity. LOH in 3p14 was demonstrated in three of seven informative cases in all tumors. LOH in 9p was demonstrated in two of five informative cases in all tumors. Two of three cases with LOH at 3p14 had a poor prognosis, one of which also had LOH at 9p. No mutation of the K-ras gene was observed in any of these tumors. These data thus indicate that p53 overexpression might distinguish atypical carcinoid tumors from typical tumors and might therefore be useful as an adjunct modality in the differential diagnosis of pulmonary carcinoid tumors. The presence of LOH at 3p14 or 9p may thus help to identify lung cancer patients with a poor prognosis.

Duodenal metastasis from primary lung cancer is extremely rare. It rarely shows any symptoms, and the prognosis for this condition is poor. We herein describe the case of a 46-year-old woman with primary lung cancer who underwent a left upper lobectomy. Severe anemia was observed about 20 days after lobectomy. Gastroduodenoscopy showed duodenal metastasis. Simultaneously, brain metastasis was also detected using magnetic resonance imaging. The patient underwent a local resection of the duodenum and a tumor resection of the brain. Postoperative irradiation of the brain metastases and systemic chemotherapy of the lung metastases were performed, and complete remission occurred. However, abdominal lymph node metastasis recurred, and the patient died 1 year after the lobectomy.