Multilocus sequence analysis of 'Candidatus Phytoplasma asteris' strain and the genome analysis of Turnip mosaic virus co-infecting oilseed rape.

AIM: Molecular characterization of a pathogenic complex infecting winter oilseed rape (Brassica napus ssp. oleifera (DC.) Metzg.) plants showing typical rape phyllody symptoms along with some atypical changes. METHODS AND RESULTS: Phytoplasma ('Candidatus Phytoplasma') presence was confirmed by PCR-RFLP and 16S rRNA gene sequencing. Phylogenetic analyses of phytoplasma amp, tufB, secY, groEL and ribosomal protein genes confirmed its affiliation to the 'Ca. P. asteris' species. However, in the amp gene encoding a specific protein crucial for insect transmission specificity, significant SNPs were found. Biological and serological tests revealed the co-infection with Turnip mosaic virus (TuMV). The phylogenetic analysis of full TuMV genome sequence, the first reported from the Balkans, classified it into the world-B phylogenetic lineage. CONCLUSIONS: A pathogenic complex consisting of 'Ca. P. asteris' and TuMV found to co-infect oilseed rape plants for the first time was molecularly characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: Rape phyllody is a serious problem in rapeseed production. The molecular information from this first multi-gene analysis of 'Ca. P. asteris' strain associated with rape phyllody as well as the first report of the complete sequence of TuMV isolate from the Balkans is a starting point for understanding the disease complexity and management.

Expansion and deletion of CTG repeats from human disease genes are determined by the direction of replication in E. coli.

Several human hereditary neurological and neurodegenerative disease genes are associated with the expansion of CTG repeats. Here we show that the frequency of genetic expansions or deletions in Escherichia coli depends on the direction of replication. Large expansions occur predominantly when the CTGs are in the leading strand template rather than the lagging strand. However, deletions are more prominent when the CTGs are in the opposite orientation. Most deletions generated products of defined size classes. Strand slippage coupled with non-classical DNA structures may account for these observations and relate to expansion-deletion mechanisms in eukaryotic chromosomes for disease genes.

Elevated 1-h plasma glucose following 75-g oral glucose load is a predictor of arterial stiffness in subjects with normal glucose tolerance.

AIMS: The study aimed to investigate arterial stiffness in subjects with normal glucose tolerance. METHODS: BMI, systolic blood pressure, fasting plasma glucose, lipid variables, ankle-brachial pressure index and brachial-ankle pulse wave velocity were measured in 2059 subjects from Takasaki city, located approximately 100 km north of Tokyo in Japan. Following a 75-g oral glucose tolerance test, only subjects with normal glucose tolerance were selected. RESULTS: One-hour post-challenge plasma glucose levels were correlated with brachial-ankle pulse wave velocity values (r = 0.340, P < 0.0001). When subjects with normal glucose tolerance were divided into three groups-group 1 (1-h plasma glucose < 8.56 mmol/l, n = 1595), group 2 (1-h plasma glucose ≥ 8.56 and < 10.17 mmol/l, n = 334) and group 3 (1-h plasma glucose ≥ 10.17 mmol/l, n = 130)-the brachial-ankle pulse wave velocity of group 3 (1473 ± 322 cm/s) was significantly higher than that of group 2 (1355 ± 252 cm/s) and brachial-ankle pulse wave velocity of group 2 was also significantly higher than that of group 1 (1275 ± 212 cm/s). CONCLUSIONS: We have identified that, in normal glucose tolerance, arterial stiffness is advanced in subjects with higher 1-h post-challenge plasma glucose in spite of the normal range for BMI, systolic blood pressure, fasting plasma glucose and lipid variables. Higher 1-h plasma glucose level is a risk factor for arterial stiffness in normal glucose tolerance.

Gene expression analysis of rheumatoid arthritis synovial lining regions by cDNA microarray combined with laser microdissection: up-regulation of inflammation-associated STAT1, IRF1, CXCL9, CXCL10, and CCL5.

OBJECTIVES: The main histological change in rheumatoid arthritis (RA) is the villous proliferation of synovial lining cells, an important source of cytokines and chemokines, which are associated with inflammation. The aim of this study was to evaluate gene expression in the microdissected synovial lining cells of RA patients, using those of osteoarthritis (OA) patients as the control. METHODS: Samples were obtained during total joint replacement from 11 RA and five OA patients. Total RNA from the synovial lining cells was derived from selected specimens by laser microdissection (LMD) for subsequent cDNA microarray analysis. In addition, the expression of significant genes was confirmed immunohistochemically. RESULTS: The 14 519 genes detected by cDNA microarray were used to compare gene expression levels in synovial lining cells from RA with those from OA patients. Cluster analysis indicated that RA cells, including low- and high-expression subgroups, and OA cells were stored in two main clusters. The molecular activity of RA was statistically consistent with its clinical and histological activity. Expression levels of signal transducer and activator of transcription 1 (STAT1), interferon regulatory factor 1 (IRF1), and the chemokines CXCL9, CXCL10, and CCL5 were statistically significantly higher in the synovium of RA than in that of OA. Immunohistochemically, the lining synovium of RA, but not that of OA, clearly expressed STAT1, IRF1, and chemokines, as was seen in microarray analysis combined with LMD. CONCLUSIONS: Our findings indicate an important role for lining synovial cells in the inflammatory and proliferative processes of RA. Further understanding of the local signalling in structural components is important in rheumatology.

Periostin, a matrix protein, is a novel biomarker for idiopathic interstitial pneumonias.

Idiopathic interstitial pneumonias (IIPs) are histopathologically classified into several types, including usual interstitial pneumonia (UIP), nonspecific interstitial pneumonia (NSIP) and cryptogenic organising pneumonia (COP). We investigated whether periostin, a matrix protein, could be used as a biomarker to assess histopathological types of IIPs. We performed immunohistochemical analyses in each histopathological type of IIP, examined serum levels of periostin in IIP patients and analysed the relationship between serum levels of periostin and the pulmonary functions in patients with idiopathic pulmonary fibrosis (IPF). Periostin was strongly expressed in lungs of UIP and fibrotic NSIP patients, whereas expression of periostin was weak in the lungs of cellular NSIP and COP patients, as well as in normal lungs. Serum levels of periostin in IPF were significantly higher than those of healthy subjects and COP patients. Furthermore, periostin levels in IPF patients were inversely correlated with their pulmonary functions. Thus, we have found that periostin is a novel component of fibrosis in IIP. Periostin may be a potential biomarker to distinguish IIP with fibrosis.

The antisense homology box: a new motif within proteins that encodes biologically active peptides.

Amphiphilic peptides approximately fifteen amino acids in length and their corresponding antisense peptides exist within protein molecules. These regions (termed antisense homology boxes) are separated by approximately fifty amino acids. Because many sense-antisense peptide pairs have been reported to recognize and bind to each other, antisense homology boxes may be involved in folding, chaperoning and oligomer formation of proteins. The antisense homology box-derived peptide CALSVDRYRAVASW, a fragment of human endothelin A receptor, proved to be a specific inhibitor of endothelin peptide (ET-1) in a smooth muscle relaxation assay. The peptide was able to block endotoxin-induced shock in rats as well. Our finding of endothelin receptor inhibitor among antisense homology box-derived peptides indicates that searching proteins for this new motif may be useful in finding biologically active peptides.

Clinicopathologic characteristics and treatment outcome of the addition of rituximab to chemotherapy for CD5-positive in comparison with CD5-negative diffuse large B-cell lymphoma.

BACKGROUND: CD5-positive (CD5+) diffuse large B-cell lymphoma (DLBCL) comprises ∼10% of DLBCLs, and it is associated with poor prognosis. The clinicopathologic characteristics and prognosis of CD5-negative (CD5-) DLBCL and CD5+ DLBCL were compared. PATIENTS AND METHODS: The subjects were 607 DLBCL patients in whom cell surface markers could be analyzed, among 930 consecutive patients registered in the Adult Lymphoma Treatment Study Group between 1998 and 2008. RESULTS: In all, 102 patients (16.8%) had CD5+ DLBCL. Compared with CD5- DLBCL, CD5+ DLBCL was more closely associated with elevated serum lactate dehydrogenase level, advanced stage, poor performance status, extranodal sites, CD10-, BCL-2+, MUM1+, and nongerminal center B-cell type. The 5-year overall survival (OS) rates of CD5+ DLBCL (n = 102) and CD5- DLBCL (n = 505) were 55% and 65%, respectively (P = 0.032), with 5-year progression-free survival (PFS) rates of 52% and 61%, respectively (P = 0.041). In the CD5+ DLBCL patients, the addition of rituximab to chemotherapy significantly improved PFS (4-year PFS, 47.4% versus 62.5%), but not OS (4-year OS, 57.8% versus 63.5%). CONCLUSIONS: For CD5+ DLBCL, the addition of rituximab to chemotherapy significantly improved the PFS, but not OS. Therefore, it is thought that a new treatment strategy is necessary for CD5+ DLBCL.

Prognostic significance of S-phase kinase-associated protein 2 and p27kip1 in patients with diffuse large B-cell lymphoma: effects of rituximab.

BACKGROUND: The F-box protein S-phase kinase-associated protein 2 (Skp2) positively regulates the G1-S transition by promoting degradation of the cyclin-dependent kinase inhibitor p27(kip1) (p27). Recent evidence has indicated an oncogenic role of Skp2 in not only carcinogenesis but also lymphomagenesis. MATERIALS AND METHODS: Clinicopathologic features and immunohistochemical expression of Skp2 and p27 were studied retrospectively in 671 patients treated with cyclophosphamide, vincristine, doxorubicin and prednisolone (CHOP) or cyclophosphamide, vincristine, doxorubicin and prednisolone plus rituximab (R-CHOP). The median follow-up periods were 43.2 months in the CHOP group (n = 425) and 24.0 months in the R-CHOP group (n = 246). RESULTS: High Skp2 or low p27 expression correlated significantly with poor overall survival (OS) and progression-free survival (P < 0.001) in both treatment groups. The prognostic value of Skp2 or p27 expression was independent of the parameters included in the International Prognostic Index by multivariate analysis. Patients with high Skp2 expression in combination with low p27 expression showed the worst survival. CONCLUSIONS: Addition of rituximab to the CHOP regimen did not provide a beneficial outcome to patients with diffuse large B-cell lymphoma with high Skp2 expression and low p27 expression. Skp2 and p27 may be useful prognostic markers in the rituximab era.

Impaired glucose tolerance, but not impaired fasting glucose, is a risk factor for early-stage atherosclerosis.

AIMS: The study aimed to investigate early-stage atherosclerosis in patients with impaired fasting glucose compared with patients with impaired glucose tolerance. METHODS: Body mass index, systolic blood pressure, fasting plasma glucose, lipid variables, ankle-brachial pressure index and brachial-ankle pulse wave velocity were measured in 2842 subjects from Takasaki city located approximately 100 km north of Tokyo in Japan. The subjects were divided into the following five groups based on a 75-g oral glucose tolerance test: (i) normal fasting plasma glucose/normal glucose tolerance group, (ii) impaired fasting glucose group, (iii) impaired glucose tolerance group, (iv) combined glucose intolerance group and (v) diabetic glucose intolerance group. RESULTS: In comparison with fasting plasma glucose levels (r = 0.269, P < 0.0001), 2-h post-challenge glucose levels were more closely correlated with pulse wave velocity values (r = 0.300, P < 0.0001). The groups with impaired glucose tolerance, combined glucose intolerance and diabetic glucose intolerance had significantly higher pulse wave velocity values compared with the groups with normal glucose tolerance and impaired fasting glucose. Multiple regression analyses showed an independent association of age, systolic blood pressures, total cholesterol, fasting and 2h plasma glucose with pulsewave velocityvalues. Furthermore, pulse wave velocity was not significantly correlated with fasting plasma glucose, but was correlated with increased 2h plasma glucose. CONCLUSIONS: Groups with impaired glucose tolerance and combined glucose intolerance had significantly higher brachio-ankle pulse wave velocity values compared with the group with normal glucose tolerance. Although the group with impaired fasting glucose showed a marginal increase in pulse wave velocity values compared with the group with normal glucose tolerance, the difference was not significant. Thus impaired glucose tolerance, but not impaired fasting glucose, is a risk factor for early-stage atherosclerosis.

Comparisons among isolates of Sweet potato feathery mottle virus using complete genomic RNA sequences.

We determined the complete or partial nucleotide sequences of eight Sweet potato feathery mottle virus (SPFMV) isolates and compared them with 12 other partial SPFMV sequences. The genome organization of the isolate Bungo (strain group C) was very different from those of isolates in the russet crack, ordinary (O), and east Africa groups. 10-O appeared to be a recombinant of isolates S and O, with a recombination site within the P1 gene. This study will help to provide a better understanding of the taxonomy and biology of SPFMV and how these features relate to virulence.

Folliculotropic mycosis fungoides with eosinophilia and CD30+ large-cell transformation: a case with a fatal outcome presenting with multifocal lesions and leonine facies.

Eosinophilia is recognized as a poor prognostic factor in patients with cutaneous T-cell lymphoma (CTCL). We report a case of folliculotropic mycosis fungoides (FMF) presenting with multiple ulcerative nodular lesions and persistent eosinophilia. Severe facial lesions resulted in a leonine appearance. On histopathological examination, nodular infiltration of large CD30+ large cells was seen. When the previous biopsy specimens were reviewed, marked folliculotropism with atypical lymphocytes was identified in previous specimens 20 years before the blastic transformation. CC chemokine receptor 3 was expressed in tumour cells, whereas CXC chemokine receptor 3 was negative. Expression of interleukin (IL)-5 was detected in a few mononuclear lymphoid cells. This case demonstrates that T helper (Th)2-polarized tumour cells may produce Th2 cytokines including IL-5, which suggests that cytokines and chemokines may contribute to persistent eosinophilia and to recruitment of eosinophils into tumour lesions in advanced FMF.

The International Prognostic Index predicts outcome in aggressive adult T-cell leukemia/lymphoma: analysis of 126 patients from the International Peripheral T-Cell Lymphoma Project.

BACKGROUND: The International Peripheral T-cell Lymphoma Project was organized to better understand the T-cell and natural killer (NK) cell lymphomas, and our task is to present the clinicopathologic correlations and therapeutic results for adult T-cell leukemia/lymphoma (ATL). PATIENTS AND METHODS: Among 1153 patients with T-cell or NK cell lymphomas, 126 patients (9.6%) with ATL were represented in this project. All were categorized as aggressive ATL, i.e. acute or lymphoma type, and 87% fell into the lymphoma type. RESULTS: The median age was 62 years and the male to female ratio was 1.2 : 1. Significant prognostic factors for overall survival (OS) by univariate analysis were the presence of B symptoms (P = 0.018), platelet count <150 x 10(9)/l (P = 0.065), and the International Prognostic Index (IPI; P = 0.019). However, multivariate analysis indicated that only the IPI was an independent predictor of OS. Combination chemotherapy including anthracyclines was given as the initial therapy in 109 of the 116 patients (94%) who received treatment, and the overall and complete response rates were 70% and 34%, respectively. However, there was no survival benefit for those receiving an anthracycline-containing regimen. CONCLUSION: Patients with aggressive ATL have a poor clinical outcome and the IPI is a useful model for predicting outcome in ATL of the lymphoma type.

Intra-operative natural sound decreases salivary amylase activity of patients undergoing inguinal hernia repair under epidural anesthesia.

BACKGROUND: The perioperative period is psychologically as well as physically stressful for patients. Although music and sound are known to reduce patients' psychological stress, a few previous studies showed an objective outcome of music. The aim of the present study was to evaluate the relaxing effect of music during epidural anesthesia, using patients' salivary amylase activity. METHODS: Thirty-two American Society of Anesthesiologists (ASA) I or II patients presenting for inguinal hernia repair under epidural anesthesia were randomly assigned to listen to sounds of a soft wind and a twitter (S group) or to have no sounds (N group). Patients' salivary amylase activity was evaluated on arrival to the operating room and at wound closure. RESULTS: Intra-operative music significantly decreased salivary amylase activity at wound closure in the S group and the activity at wound closure of the S group was significantly smaller than that of the N group. CONCLUSION: Intra-operative natural sound significantly decreased salivary amylase activity of patients undergoing inguinal hernia repair under epidural anesthesia.

Long pulse production of high current D(-) ion beams in the JT-60 negative ion source.

The first long pulse production of high power D(-) ion beams has been demonstrated in the JT-60 U negative ion sources, each of which was designed to produce 22 A, 500 keV D(-) ion beams. Voltage holding capability and the grid power loading were examined for long pulse production of high power D(-) ion beams. From the correlation between voltage holding and the light intensity of cathodoluminescence from the Fiber Reinforced Plastic insulators, the acceleration voltage for stable voltage holding capability was found to be less than 320-340 kV where the light was sufficiently suppressed. By tuning the extraction voltage, the grid power loadings in the ion sources were decreased to the allowable levels for long pulse injection without a significant reduction of the beam power. After tuning the acceleration and extraction voltages, D(-) ion beams of 12.5 and 9.8 A were produced at 340 keV with cesium seeding at a rate of approximately 14 microg/s into the ion sources. The pulse duration of these D(-) ion beams was extended step by step, and then was successfully extended up to 18 s without degradation of the negative ion production. The D(-) ion beams were neutralized to yield 3.6 MW D(0) beams by a gas cell, and then injected into the JT-60 U plasma. Further, a slight reduction of D(-) ion beam power allowed the longer injection duration of 21 s at a D(0) beam power of 3.2 MW. The success in the long pulse production of a high power D(-) ion beam shows that negative ion beams can be produced during a few tens of seconds without degradations of negative ion production and the voltage holding in a large Cs-seeded negative ion source.

Activation of RHOA-VAV1 signaling in angioimmunoblastic T-cell lymphoma.

Somatic G17V RHOA mutations were found in 50-70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high-throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1-STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), whereas none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in VAV1-STAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1-STAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA-VAV1 axis may provide a new therapeutic target in AITL.

Integrating genomic alterations in diffuse large B-cell lymphoma identifies new relevant pathways and potential therapeutic targets.

Genome studies of diffuse large B-cell lymphoma (DLBCL) have revealed a large number of somatic mutations and structural alterations. However, the clinical significance of these alterations is still not well defined. In this study, we have integrated the analysis of targeted next-generation sequencing of 106 genes and genomic copy number alterations (CNA) in 150 DLBCL. The clinically significant findings were validated in an independent cohort of 111 patients. Germinal center B-cell and activated B-cell DLBCL had a differential profile of mutations, altered pathogenic pathways and CNA. Mutations in genes of the NOTCH pathway and tumor suppressor genes (TP53/CDKN2A), but not individual genes, conferred an unfavorable prognosis, confirmed in the independent validation cohort. A gene expression profiling analysis showed that tumors with NOTCH pathway mutations had a significant modulation of downstream target genes, emphasizing the relevance of this pathway in DLBCL. An in silico drug discovery analysis recognized 69 (46%) cases carrying at least one genomic alteration considered a potential target of drug response according to early clinical trials or preclinical assays in DLBCL or other lymphomas. In conclusion, this study identifies relevant pathways and mutated genes in DLBCL and recognizes potential targets for new intervention strategies.

The 3' ends of tRNA-derived short interspersed repetitive elements are derived from the 3' ends of long interspersed repetitive elements.

Short interspersed repetitive elements (SINEs) are a type of retroposon, being members of a class of informational molecules that are amplified via cDNA intermediates and flow back into the host genome. In contrast to retroviruses and retrotransposons, SINEs do not encode the enzymes required for their amplification, such as reverse transcriptases, so they are presumed to borrow these enzymes from other sources. In the present study, we isolated a family of long interspersed repetitive elements (LINEs) from the turtle genome. The sequence of this family was found to be very similar to those of the avian CR1 family. To our surprise, the sequence at the 3' end of the LINE in the turtle genome was nearly identical to that of a family of tortoise SINEs. Since CR1-like LINEs are widespread in birds and in many other reptiles, including the turtle, and since the tortoise SINEs are only found in vertical-necked turtles, it seems possible that the sequence at the 3' end of the tortoise SINEs might have been generated by recombination with the CR1-like LINE in a common ancestor of vertical-necked turtles, after the divergence of side-necked turtles. We extended our observations to show that the 3'-end sequences of families of several tRNA-derived SINEs, such as the salmonid HpaI family, the tobacco TS family, and the salmon SmaI family, might have originated from the respective LINEs. Since it appears reasonable that the recognition sites of LINEs for reverse transcriptase are located within their 3'-end sequences, these results provide the basis for a general scheme for the mechanism by which SINEs might acquire retropositional activity. We propose here that tRNA-derived SINEs might have been generated by a recombination event in which a strong-stop DNA with a primer tRNA, which is an intermediate in the replication of certain retroviruses and long terminal repeat retrotransposons, was directly integrated at the 3' end of a LINE.

Identification of cell-type-specific mutations in nodal T-cell lymphomas.

Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations.

Low-grade follicular lymphoma with t(14;18) presents a homogeneous disease entity otherwise the rest comprises minor groups of heterogeneous disease entities with Bcl2 amplification, Bcl6 translocation or other gene aberrances.

Follicular lymphomas (FL) are morphologically classified into grades 1, 2, 3a and 3b by the World Health Organization. Bcl2, Bcl6 and CD10 are phenotypic markers of FL while the Bcl2 t(14;18) and Bcl6 t(3q27) gene translocations are common genetic changes. However, to date, there has been no integrated analysis based on phenotype, grade and genotype from large numbers of FL cases. We graded 261 cases of FL and determined their phenotypes and gene alterations. According to the antigen markers and gene alterations of 147 cases, we classified FL into typical and the others types. The typical group, which includes 69% cases of FL, is characterized by low histological grade (grade 1, 2), coexpression of BCL2 and CD10 and Bcl2 gene translocation. The rest comprises a small part of low-grade FL without Bcl2 gene translocation and high-grade (grade 3a, 3b) FL. These FLs include some heterogeneous disease entities. They are characterized by high histological grade (87%), no definite expression of BCL2 or CD10 and several kinds of gene aberrances including Bcl2 translocation, Bcl6 translocation, Bcl2 amplification or other unknown gene abnormality. Our findings indicate that typical FL presents a homogeneous disease entity whereas the rest comprises heterogeneous diseases entities.