Heterologous gene expression system for the production of hydrolyzable tannin intermediates in herbaceous model plants.

Aluminum toxicity is the main factor limiting the elongation of plant roots in acidic soil. The tree species Eucalyptus camaldulensis is considerably more resistant to aluminum than herbaceous model plants and crops. Hydrolyzable tannins (HTs) accumulating in E. camaldulensis roots can bind and detoxify the aluminum taken up by the roots. However, in herbaceous model plants, HTs do not accumulate and the genes involved in the HT biosynthetic pathway are largely unknown. The aim of this study was to establish a method for reconstituting the HT biosynthetic pathway in the HT non-accumulating model plant Nicotiana benthamiana. Four E. camaldulensis enzymes were transiently expressed in N. benthamiana leaves via Agrobacterium tumefaciens-mediated transformation. These enzymes included dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDH2 and EcDQD/SDH3), which catalyze the synthesis of gallic acid, the first intermediate of the HT biosynthetic pathway that branches off from the shikimate pathway. The others were UDP-glycosyltransferases (UGT84A25 and UGT84A26), which catalyze the conversion of gallic acid to ß-glucogallin, the second intermediate. The co-expression of the EcDQD/SDHs in transgenic N. benthamiana leaf regions promoted the synthesis of gallic acid. Moreover, the co-expression of the UGT84As in addition to the EcDQD/SDHs resulted in the biosynthesis of ß-glucogallin, the universal metabolic precursor of HTs. Thus, we successfully reconstituted a portion of the HT biosynthetic pathway in HT non-accumulating N. benthamiana plants. This heterologous gene expression system will be useful for co-expressing candidate genes involved in downstream reactions in the HT biosynthetic pathway and for clarifying their in planta functions.

Different photorespiratory mechanisms in conifer leaves, where peroxisomes have intrinsically low catalase activity.

Photorespiration is an essential metabolic mechanism associated with photosynthesis; however, little is known about the photorespiratory pathway of conifer gymnosperms. Metabolite analyses of the leaves of 27 tree species showed that the mean glycerate content in conifer leaves was lower than that in angiosperm leaves. We performed experiments where [13 C]-serine was fed to detached shoots of a conifer (Cryptomeria japonica), via the transpiration stream, and compared the labeling patterns of photorespiratory metabolites with those of an angiosperm tree (Populus nigra), because glycerate is produced from serine via hydroxypyruvate in peroxisomes. In P. nigra, hydroxypyruvate, glycerate and glycine were labeled with 13 C, whereas in C. japonica, glycolate and a non-canonical photorespiratory metabolite, formate, were also labeled, suggesting that an H2 O2 -mediated non-enzymatic decarboxylation (NED) reaction occurs in C. japonica. We analyzed changes in the metabolite contents of leaves kept in the dark and leaves exposed to illuminated photorespiration-promoting conditions: a positive relationship between formate and serine levels in C. japonica implied that the active C1 -metabolism pathway synthesizes serine from formate. Leaf gas exchange analyses revealed that CO2 produced through NED was recaptured by chloroplasts. Database analysis of the peroxisomal targeting signal motifs of an H2 O2 -scavenging enzyme, catalase, derived from various species, including nine coniferous species, as well as analyses of peroxisomal fractions isolated from C. japonica and P. nigra leaves indicated that conifer peroxisomes had less catalase activity. These results suggest that NED and the subsequent C1 metabolism are involved in the photorespiratory pathway of conifer leaves, where peroxisomes have intrinsically low catalase activity.

[Hemorrhagic Colon Cancer with Left Atrial Thrombus Formation after Anticoagulant Therapy Discontinuation-A Case Report].

The patient was a 72-year-old female. She had been taking rivaroxaban for chronic atrial fibrillation; however, she stopped taking it due to anemia and was hospitalized urgently. A contrast-enhanced computed tomography(CT)scan showed a 30 mm mass in the ascending colon, and a colonoscopy revealed ascending colon cancer(cT3, cN0, cM0, cStage Ⅱa). The tumor was hemorrhagic and was thought to have caused the anemia. On day 6 of hospitalization, another contrast- enhanced CT scan showed a poorly contrast-enhanced area in the left atrium, and transesophageal echocardiography revealed 2 left atrial thrombi(27 mm and 17 mm). Since early induction of anticoagulation therapy was considered, an emergency open right colectomy was performed to remove the cause of the bleeding. Intravenous heparin therapy was started the day after surgery and was switched to oral apixaban therapy on the fourth postoperative day. The postoperative course was good, and she was discharged home on the 17th postoperative day. This patient had conflicting clinical problems simultaneously; however, immediate decision-making and initiation of treatment were effective.

Dehydroquinate dehydratase/shikimate dehydrogenases involved in gallate biosynthesis of the aluminum-tolerant tree species Eucalyptus camaldulensis.

MAIN CONCLUSION: Eucalyptus camaldulensis EcDQD/SDH2 and 3 combine gallate formation, dehydroquinate dehydratase, and shikimate dehydrogenase activities. They are candidates for providing the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B. The tree species Eucalyptus camaldulensis shows exceptionally high tolerance against aluminum, a widespread toxic metal in acidic soils. In the roots of E. camaldulensis, aluminum is detoxified via the complexation with oenothein B, a hydrolyzable tannin. In our approach to elucidate the biosynthesis of oenothein B, we here report on the identification of E. camaldulensis enzymes that catalyze the formation of gallate, which is the phenolic constituent of hydrolyzable tannins. By systematical screening of E. camaldulensis dehydroquinate dehydratase/shikimate dehydrogenases (EcDQD/SDHs), we found two enzymes, EcDQD/SDH2 and 3, catalyzing the NADP+-dependent oxidation of 3-dehydroshikimate to produce gallate. Based on extensive in vitro assays using recombinant EcDQD/SDH2 and 3 enzymes, we present for the first time a detailed characterization of the enzymatic gallate formation activity, including the cofactor preferences, pH optima, and kinetic constants. Sequence analyses and structure modeling suggest the gallate formation activity of EcDQD/SDHs is based on the reorientation of 3-dehydroshikimate in the catalytic center, which facilitates the proton abstraction from the C5 position. Additionally, EcDQD/SDH2 and 3 maintain DQD and SDH activities, resulting in a 3-dehydroshikimate supply for gallate formation. In E. camaldulensis, EcDQD/SDH2 and 3 are co-expressed with UGT84A25a/b and UGT84A26a/b involved in hydrolyzable tannin biosynthesis. We further identified EcDQD/SDH1 as a "classical" bifunctional plant shikimate pathway enzyme and EcDQD/SDH4a/b as functional quinate dehydrogenases of the NAD+/NADH-dependent clade. Our data indicate that in E. camaldulensis the enzymes EcDQD/SDH2 and 3 provide the essential gallate for the biosynthesis of the aluminum-detoxifying metabolite oenothein B.

[Locally Advanced Rectal Cancer Presenting with Perforation That Was Successfully Resected after Preoperative Chemotherapy with mFOLFOX6 plus Panitumumab].

We report a case of locally advanced rectal cancer presenting with perforation that was successfully resected after preoperative chemotherapy. A 66-year-old woman visited our emergency room complaining of lower abdominal pain. Abdominal CT showed a rectal tumor with fluid collection and free air in the pelvis. The patient was diagnosed with panperitonitis secondary to cancerous perforation and underwent sigmoid colostomy. A biopsy specimen of the rectal tumor showed well-differentiated tubular adenocarcinoma and wild-type RAS. After 8 courses of mFOLFOX6 plus panitumumab, the tumor shrank remarkably, and radical surgery(low anterior resection with D3 lymph node dissection)was performed. Microscopic examination of the resected specimen showed that almost half of the tumor cells were replaced by histiocytes and necrotic tissue. Preoperative chemotherapy with panitumumab may be an effective treatment for RAS wild-type locally advanced colon cancer, even if the primary tumor develops perforation.

[Nephrotic Syndrome Induced by Ramucirumab for Metastatic Rectal Cancer].

We report a case of nephrotic syndrome induced by ramucirumab for metastatic rectal cancer. A 48-year old woman who had received FOLFIRI plus ramucirumab for rectal cancer with liver, lung, and bone metastases presented to our hospital with edema and increased body weight. Laboratory studies showed hypoalbuminemia and severe proteinuria, and the patient was diagnosed with nephrotic syndrome. After administration of an angiotensin II receptor blocker and thiazide diuretic, her symptoms disappeared and serum albumin level normalized. To the best of our knowledge, this is the first detailed report of nephrotic syndrome related to ramucirumab. This case serves to emphasize that the possibility of nephrotic syndrome should be considered during anti-vascular endothelial growth factor therapy.

Identification of UDP glucosyltransferases from the aluminum-resistant tree Eucalyptus camaldulensis forming ß-glucogallin, the precursor of hydrolyzable tannins.

In the highly aluminum-resistant tree Eucalyptus camaldulensis, hydrolyzable tannins are proposed to play a role in internal detoxification of aluminum, which is a major factor inhibiting plant growth on acid soils. To understand and modulate the molecular mechanisms of aluminum detoxification by hydrolyzable tannins, the biosynthetic genes need to be identified. In this study, we identified and characterized genes encoding UDP-glucose:gallate glucosyltransferase, which catalyzes the formation of 1-O-galloyl-ß-d-glucose (ß-glucogallin), the precursor of hydrolyzable tannins. By homology-based cloning, seven full-length candidate cDNAs were isolated from E. camaldulensis and expressed in Escherichia coli as recombinant N-terminal His-tagged proteins. Phylogenetic analysis classified four of these as UDP glycosyltransferase (UGT) 84A subfamily proteins (UGT84A25a, -b, UGT84A26a, -b) and the other three as UGT84J subfamily proteins (UGT84J3, -4, -5). In vitro enzyme assays showed that the UGT84A proteins catalyzed esterification of UDP-glucose and gallic acid to form 1-O-galloyl-ß-d-glucose, whereas the UGT84J proteins were inactive. Further analyses with UGT84A25a and -26a indicated that they also formed 1-O-glucose esters of other structurally related hydroxybenzoic and hydroxycinnamic acids with a preference for hydroxybenzoic acids. The UGT84A genes were expressed in leaves, stems, and roots of E. camaldulensis, regardless of aluminum stress. Taken together, our results suggest that the UGT84A subfamily enzymes of E. camaldulensis are responsible for constitutive production of 1-O-galloyl-ß-d-glucose, which is the first step of hydrolyzable tannin biosynthesis.

Heavy-ion beam mutagenesis of the ectomycorrhizal agaricomycete Tricholoma matsutake that produces the prized mushroom "matsutake" in conifer forests.

Tricholoma matsutake is an ectomycorrhizal agaricomycete that produces the prized mushroom "matsutake" in Pinaceae forests. Currently, there are no available cultivars or cultivation methods that produce fruiting bodies. Heavy-ion beams, which induce mutations through double-stranded DNA breaks, have been used widely for plant breeding. In the present study, we examined whether heavy-ion beams could be useful in isolating T. matsutake mutants. An argon-ion beam gave a suitable lethality curve in relation to irradiation doses, accelerating killing at 100-150 Gy. Argon-ion beam irradiation of the agar plate cultures yielded several transient mutants whose colony morphologies differed from that of the wild-type strain at the first screening, but which did not persist following culture transfer. It also generated a mutant whose phenotype remained stable after repeated culture transfers. The stable pleiotropic mutant not only exhibited a different colony morphology to the wild type, but also showed increased degradation of dye-linked water-insoluble amylose and cellulose substrates. Thus, heavy-ion beams may be useful for isolating mutants of T. matsutake, although precautions may be required to maintain the mutants, without phenotypic reversion, during repetitive culture of their mycelia.

miR-125b-1 and miR-378a are predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer.

Many clinical trials of peptide vaccines have been conducted. However, these vaccines have provided clinical benefits in only a small fraction of patients. The purpose of the present study was to explore microRNAs (miRNAs) as novel predictive biomarkers for the efficacy of vaccine treatment against colorectal cancer. First, we carried out microarray analysis of pretreatment cancer tissues in a phase I study, in which peptide vaccines alone were given. Candidate miRNAs were selected by comparison of the better prognosis group with the poorer prognosis group. Next, we conducted microarray analysis of cancer tissues in a phase II study, in which peptide vaccines combined with chemotherapy were given. Candidate miRNAs were further selected by a similar comparison of prognosis. Subsequently, we carried out reverse-transcription PCR analysis of phase II cases, separating cancer tissues into cancer cells and stromal tissue using laser capture microdissection. Treatment effect in relation to overall survival (OS) and miRNA expression was analyzed. Three miRNA predictors were negatively associated with OS: miR-125b-1 in cancer cells (P = 0.040), and miR-378a in both cancer cells (P = 0.009) and stromal cells (P < 0.001). Multivariate analysis showed that expression of miR-378a in stromal cells was the best among the three predictors (HR, 2.730; 95% CI, 1.027-7.585; P = 0.044). In conclusion, miR-125b-1 and miR-378a expression might be considered as novel biomarkers to predict the efficacy of vaccine treatment against colorectal cancer.

A phase ΙI study of five peptides combination with oxaliplatin-based chemotherapy as a first-line therapy for advanced colorectal cancer (FXV study).

BACKGROUND: We previously conducted a phase I trial for advanced colorectal cancer (CRC) using five HLA-A*2402-restricted peptides, three derived from oncoantigens and two from vascular endothelial growth factor (VEGF) receptors, and confirmed safety and immunological responses. To evaluate clinical benefits of cancer vaccination treatment, we conducted a phase II trial using the same peptides in combination with oxaliplatin-based chemotherapy as a first-line therapy. METHODS: The primary objective of the study was the response rates (RR). Progression free survival (PFS), overall survival (OS), and immunological parameters were evaluated as secondary objective. The planned sample size was more than 40 patients for both HLA2402-matched and -unmatched groups. All patients received a cocktail of five peptides (3 mg each) mixed with 1.5 ml of IFA which was subcutaneously administered weekly for the first 12 weeks followed by biweekly administration. Presence or absence of the HLA-A*2402 genotype were used for classification of patients into two groups. RESULTS: Between February 2009 and November 2012, ninety-six chemotherapy naïve CRC patients were enrolled under the masking of their HLA-A status. Ninety-three patients received mFOLFOX6 and three received XELOX. Bevacizumab was added in five patients. RR was 62.0% and 60.9% in the HLA-A*2402-matched and -unmatched groups, respectively (p=0.910). The median OS was 20.7 months in the HLA-A*2402-matched group and 24.0 months in the unmatched group (log-rank, p=0.489). In subgroup with a neutrophil/lymphocyte ratio (NLR) of <3.0, patients in the HLA-matched group did not survive significantly longer than those in the unmatched group (log-rank, p=0.289) but showed a delayed response. CONCLUSIONS: Although no significance was observed for planned statistical efficacy endpoints, a delayed response was observed in subgroup with a NLR of <3.0. Biomarkers such as NLR might be useful for selecting patients with a better treatment outcome by the vaccination. TRIAL REGISTRATION: Trial registration: UMIN000001791.

Identification of a hydrolyzable tannin, oenothein B, as an aluminum-detoxifying ligand in a highly aluminum-resistant tree, Eucalyptus camaldulensis.

Eucalyptus camaldulensis is a tree species in the Myrtaceae that exhibits extremely high resistance to aluminum (Al). To explore a novel mechanism of Al resistance in plants, we examined the Al-binding ligands in roots and their role in Al resistance of E. camaldulensis. We identified a novel type of Al-binding ligand, oenothein B, which is a dimeric hydrolyzable tannin with many adjacent phenolic hydroxyl groups. Oenothein B was isolated from root extracts of E. camaldulensis by reverse-phase high-performance liquid chromatography and identified by nuclear magnetic resonance and mass spectrometry analyses. Oenothein B formed water-soluble or -insoluble complexes with Al depending on the ratio of oenothein B to Al and could bind at least four Al ions per molecule. In a bioassay using Arabidopsis (Arabidopsis thaliana), Al-induced inhibition of root elongation was completely alleviated by treatment with exogenous oenothein B, which indicated the capability of oenothein B to detoxify Al. In roots of E. camaldulensis, Al exposure enhanced the accumulation of oenothein B, especially in EDTA-extractable forms, which likely formed complexes with Al. Oenothein B was localized mostly in the root symplast, in which a considerable amount of Al accumulated. In contrast, oenothein B was not detected in three Al-sensitive species, comprising the Myrtaceae tree Melaleuca bracteata, Populus nigra, and Arabidopsis. Oenothein B content in roots of five tree species was correlated with their Al resistance. Taken together, these results suggest that internal detoxification of Al by the formation of complexes with oenothein B in roots likely contributes to the high Al resistance of E. camaldulensis.

The Effects of Percutaneous Endoscopic Gastrostomy on Quality of Life in Patients With Dementia.

BACKGROUND: To examine the effects of percutaneous endoscopic gastrostomy (PEG) on quality of life (QOL) in patients with dementia. METHODS: We retrospectively included 53 Japanese community and tertiary hospitals to investigate the relationship between the newly developed PEG and consecutive dementia patients with swallowing difficulty between Jan 1st 2006 and Dec 31st 2008. We set improvements in 1) the level of independent living, 2) pneumonia, 3) peroral intake as outcome measures of QOL and explored the factors associated with these improvements. RESULTS: Till October 31st 2010, 1,353 patients with Alzheimer's dementia (33.1%), vascular dementia (61.7%), dementia with Lewy body disease (2.0%), Pick disease (0.6%) and others were followed-up for a median of 847 days (mean 805 ± 542 days). A total of 509 deaths were observed (mortality 59%) in full-followed patients. After multivariate adjustments, improvement in the level of independent living was observed in milder dementia, or those who can live independently with someone, compared with advanced dementia, characterized by those who need care by someone: Odds Ratio (OR), 3.90, 95% confidence interval (95%CI), 1.59 - 9.39, P = 0.003. Similarly, improvement of peroral intake was noticed in milder dementia: OR, 2.69, 95%CI, 1.17 - 6.17, P = 0.02. Such significant associations were not observed in improvement of pneumonia. CONCLUSIONS: These results suggest that improvement of QOL after PEG insertion may be expected more in milder dementia than in advanced dementia.