Plastid genome characterisation in Brassica and Brassicaceae using a new set of nine SSRs.

We report a new set of nine primer pairs specifically developed for amplification of Brassica plastid SSR markers. The wide utility of these markers is demonstrated for haplotype identification and detection of polymorphism in B. napus, B. nigra, B. oleracea, B. rapa and in related genera Arabidopsis, Camelina, Raphanus and Sinapis. Eleven gene regions (ndhB-rps7 spacer, rbcL-accD spacer, rpl16 intron, rps16 intron, atpB-rbcL spacer, trnE-trnT spacer, trnL intron, trnL-trnF spacer, trnM-atpE spacer, trnR-rpoC2 spacer, ycf3-psaA spacer) were sequenced from a range of Brassica and related genera for SSR detection and primer design. Other sequences were obtained from GenBank/EMBL. Eight out of nine selected SSR loci showed polymorphism when amplified using the new primers and a combined analysis detected variation within and between Brassica species, with the number of alleles detected per locus ranging from 5 (loci MF-6, MF-1) to 11 (locus MF-7). The combined SSR data were used in a neighbour-joining analysis (SMM, D (DM) distances) to group the samples based on the presence and absence of alleles. The analysis was generally able to separate plastid types into taxon-specific groups. Multi-allelic haplotypes were plotted onto the neighbour joining tree. A total number of 28 haplotypes were detected and these differentiated 22 of the 41 accessions screened from all other accessions. None of these haplotypes was shared by more than one species and some were not characteristic of their predicted type. We interpret our results with respect to taxon differentiation, hybridisation and introgression patterns relating to the 'Triangle of U'.

Benthic macroinvertebrates assemblages and macrohabitat connectivity in Mediterranean-climate streams of northern California

Abstract. Drought leads to a loss of longitudinal and lateral hydrologic connectivity, which causes direct or indirect changes in stream ecosystem properties. Changes in macrohabitat availability from a rifflepool sequence to isolated pools are among the most conspicuous consequences of connectivity loss. Macroinvertebrate assemblages were compared among 3 distinct stream macrohabitats (riffles [R], pools connected to riffles [Pc], disconnected pools [Pd]) of 19 Mediterranean-climate sites in northern California to examine the influence of loss of habitat resulting from drought disturbance. At the time of sampling, 10 sites were perennial and included R and Pc macrohabitats, whereas 9 sites were intermittent and included only Pd macrohabitats. Taxa richness was more variable in Pd, and taxa richness was significantly lower in Pd than in Pc but not R. These results suggested a decline in richness between Pc and Pd that might be associated with loss of connectivity. Lower Ephemeroptera, Plecoptera, and Trichoptera (EPT) richness relative to Odonata, Coleoptera, and Heteroptera (OCH) richness was observed for Pd than R and Pc macrohabitats. Family composition was more similar between R and Pc than between R or Pc and Pd macrohabitats. This similarity may be associated with greater connectivity between R and Pc macrohabitats. Correspondence analysis indicated that macroinvertebrate composition changed along a gradient from R to Pc and Pd that was related to a perennialintermittent gradient across sites. High variability among macroinvertebrate assemblages in Pd could have been related to variability in the duration of intermittency. In cluster analysis, macroinvertebrate assemblages were grouped by macrohabitat first and then by site, suggesting that the macrohabitat filter had a greater influence on macroinvertebrate assemblages than did local site characteristics. Few taxa were found exclusively in Pc, and this macrohabitat shared numerous taxa with R and Pd, indicating that Pc may act as a bridge between R and Pd during drought. Drought is regarded as a ramp disturbance, but our results suggest that the response of macroinvertebrate assemblages to the loss of hydrological connectivity among macrohabitats is gradual, at least in Mediterranean-climate streams where drying is gradual. However, the changes may be more dramatic in arid and semiarid streams or in Mediterranean-climate streams if drying is rapid.

Case complexity and clinical outcome in diabetes mellitus. A prospective study using the INTERMED.

The aim of this study was to assess a population of patients with diabetes mellitus by means of the INTERMED, a classification system for case complexity integrating biological, psychosocial and health care related aspects of disease. The main hypothesis was that the INTERMED would identify distinct clusters of patients with different degrees of case complexity and different clinical outcomes. Patients (n=61) referred to a tertiary reference care centre were evaluated with the INTERMED and followed 9 months for HbA1c values and 6 months for health care utilisation. Cluster analysis revealed two clusters: cluster 1 (62%) consisting of complex patients with high INTERMED scores and cluster 2 (38%) consisting of less complex patients with lower INTERMED. Cluster 1 patients showed significantly higher HbA1c values and a tendency for increased health care utilisation. Total INTERMED scores were significantly related to HbA1c and explained 21% of its variance. In conclusion, different clusters of patients with different degrees of case complexity were identified by the INTERMED, allowing the detection of highly complex patients at risk for poor diabetes control. The INTERMED therefore provides an objective basis for clinical and scientific progress in diabetes mellitus. Ongoing intervention studies will have to confirm these preliminary data and to evaluate if management strategies based on the INTERMED profiles will improve outcomes.

Analytical Measures of Learners' Written Interlanguage

This paper aims at reconsidering some analytical measures to best encapsulate the interlanguage, in writing, of young beginner learners of English as a foreign language in the light of previous and work-in-progress research conducted within the BAF project, and in particular, whether clause and sentence length should be best viewed as a fluency or syntactic complexity measusre or as part of a different construct. In the light of a factor analysis (Navés, forthcoming) and multivariate and correlation studies (Navés et al. 2003, Navés, 2006, Torres et al. 2006) it becomes clear that the relationship between different analytical measures is also dependent on learner¿s cognitive maturity (age) and proficiency (amount of instruction). Finally, clause and sentence length should not be viewed as either a fluency or sytactic complexity measure but as part of a different construct. It is concluded that further research using regression analysis and cluster analysis is neeed in order to identify and validate the constructs of the writing components and their measurements.

Structural assessment of single amino acid mutations: application to TP53 function.

Single amino acid substitution is the type of protein alteration most related to human diseases. Current studies seek primarily to distinguish neutral mutations from harmful ones. Very few methods offer an explanation of the final prediction result in terms of the probable structural or functional effect on the protein. In this study, we describe the use of three novel parameters to identify experimentally-verified critical residues of the TP53 protein (p53). The first two parameters make use of a surface clustering method to calculate the protein surface area of highly conserved regions or regions with high nonlocal atomic interaction energy (ANOLEA) score. These parameters help identify important functional regions on the surface of a protein. The last parameter involves the use of a new method for pseudobinding free-energy estimation to specifically probe the importance of residue side-chains to the stability of protein fold. A decision tree was designed to optimally combine these three parameters. The result was compared to the functional data stored in the International Agency for Research on Cancer (IARC) TP53 mutation database. The final prediction achieved a prediction accuracy of 70% and a Matthews correlation coefficient of 0.45. It also showed a high specificity of 91.8%. Mutations in the 85 correctly identified important residues represented 81.7% of the total mutations recorded in the database. In addition, the method was able to correctly assign a probable functional or structural role to the residues. Such information could be critical for the interpretation and prediction of the effect of missense mutations, as it not only provided the fundamental explanation of the observed effect, but also helped design the most appropriate laboratory experiment to verify the prediction results.

Improved multiple-locus variable-number tandem-repeat assay for Staphylococcus aureus genotyping, providing a highly informative technique together with strong phylogenetic value.

We describe an improved multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) scheme for genotyping Staphylococcus aureus. We compare its performance to those of multilocus sequence typing (MLST) and spa typing in a survey of 309 strains. This collection includes 87 epidemic methicillin-resistant S. aureus (MRSA) strains of the Harmony collection, 75 clinical strains representing the major MLST clonal complexes (CCs) (50 methicillin-sensitive S. aureus [MSSA] and 25 MRSA), 135 nasal carriage strains (133 MSSA and 2 MRSA), and 13 published S. aureus genome sequences. The results show excellent concordance between the techniques' results and demonstrate that the discriminatory power of MLVA is higher than those of both MLST and spa typing. Two hundred forty-two genotypes are discriminated with 14 VNTR loci (diversity index, 0.9965; 95% confidence interval, 0.9947 to 0.9984). Using a cutoff value of 45%, 21 clusters are observed, corresponding to the CCs previously defined by MLST. The variability of the different tandem repeats allows epidemiological studies, as well as follow-up of the evolution of CCs and the identification of potential ancestors. The 14 loci can conveniently be analyzed in two steps, based upon a first-line simplified assay comprising a subset of 10 loci (panel 1) and a second subset of 4 loci (panel 2) that provides higher resolution when needed. In conclusion, the MLVA scheme proposed here, in combination with available on-line genotyping databases (including http://mlva.u-psud.fr/), multiplexing, and automatic sizing, can provide a basis for almost-real-time large-scale population monitoring of S. aureus.

Debris flows as a factor of hillslope evolution controlled by a continuous or a pulse process?

Flood effectiveness observations imply that two families of processes describe the formation of debris flow volume. One is related to the rainfall?erosion relationship, and can be seen as a gradual process, and one is related to additional geological/geotechnical events, those named hereafter extraordinary events. In order to discuss the hypothesis of coexistence of two modes of volume formation, some methodologies are applied. Firstly, classical approaches consisting in relating volume to catchments characteristics are considered. These approaches raise questions about the quality of the data rather than providing answers concerning the controlling processes. Secondly, we consider statistical approaches (cumulative number of events distribution and cluster analysis) and these suggest the possibility of having two distinct families of processes. However the quantitative evaluation of the threshold differs from the one that could be obtained from the first approach, but they all agree in the sense of the coexistence of two families of events. Thirdly, a conceptual model is built exploring how and why debris flow volume in alpine catchments changes with time. Depending on the initial condition (sediment production), the model shows that large debris flows (i.e. with important volume) are observed in the beginning period, before a steady-state is reached. During this second period debris flow volume such as is observed in the beginning period is not observed again. Integrating the results of the three approaches, two case studies are presented showing: (1) the possibility to observe in a catchment large volumes that will never happen again due to a drastic decrease in the sediment availability, supporting its difference from gradual erosion processes; (2) that following a rejuvenation of the sediment storage (by a rock avalanche) the magnitude?frequency relationship of a torrent can be differentiated into two phases, the beginning one with large and frequent debris flow and a later one with debris flow less intense and frequent, supporting the results of the conceptual model. Although the results obtained cannot identify a clear threshold between the two families of processes, they show that some debris flows can be seen as pulse of sediment differing from that expected from gradual erosion.

Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data.

In order to contribute to the debate about southern glacial refugia used by temperate species and more northern refugia used by boreal or cold-temperate species, we examined the phylogeography of a widespread snake species (Vipera berus) inhabiting Europe up to the Arctic Circle. The analysis of the mitochondrial DNA (mtDNA) sequence variation in 1043 bp of the cytochrome b gene and in 918 bp of the noncoding control region was performed with phylogenetic approaches. Our results suggest that both the duplicated control region and cytochrome b evolve at a similar rate in this species. Phylogenetic analysis showed that V. berus is divided into three major mitochondrial lineages, probably resulting from an Italian, a Balkan and a Northern (from France to Russia) refugial area in Eastern Europe, near the Carpathian Mountains. In addition, the Northern clade presents an important substructure, suggesting two sequential colonization events in Europe. First, the continent was colonized from the three main refugial areas mentioned above during the Lower-Mid Pleistocene. Second, recolonization of most of Europe most likely originated from several refugia located outside of the Mediterranean peninsulas (Carpathian region, east of the Carpathians, France and possibly Hungary) during the Mid-Late Pleistocene, while populations within the Italian and Balkan Peninsulas fluctuated only slightly in distribution range, with larger lowland populations during glacial times and with refugial mountain populations during interglacials, as in the present time. The phylogeographical structure revealed in our study suggests complex recolonization dynamics of the European continent by V. berus, characterized by latitudinal as well as altitudinal range shifts, driven by both climatic changes and competition with related species.

Isoenzymatic variability in wild potatoes

Two species of wild potato Solanum commersonii, subspecies commersonii and malmeanum, and S. chacoense, subspecies muelleri occur in southern Brazil. Their rusticity and easy adaptation to extreme climatic conditions make them valuable for breeding programs. The objective of this work was to analyze the isoenzymatic variability of 113 clones of wild potato subspecies. They were collected and maintained at Embrapa-Centro de Pesquisa Agropecuária de Clima Temperado, at Pelotas, RS, Brazil. Enzymes involved in energetic (group I) or in peripherical (group II) metabolism constituted the material used. Polyacrylamide horizontal gel electrophoresis was used to analyze peroxidase, aspartate transaminase, phosphoglucomutase and isocitrate dehydrogenase isoenzymes. Solanum spp. has considerable genetic variability for isoenzymatic patterns. Cluster analysis classified the clones into 51 subgroups, based on electrophoretic variants of group I enzymes, and into 89, when group II enzyme variants were added. Genotypic differentiation of S. chacoense muelleri in relation to S. commersonii commersonii and S. commersonii malmeanum is evident when expressed through similarity and cluster analysis.

Genetic diversity and relationship in American and African oil palm as revealed by RFLP and AFLP molecular markers

The objective of this work was to evaluate the genetic diversity, its organization and the genetic relationships within oil palm (Elaeis oleifera (Kunth) Cortés, from America, and E. guineensis (Jacq.), from Africa) germplasm using Restriction Fragment Length Polymorphism (RFLP) and Amplified Fragment Length Polymorphism (AFLP). In complement to a previous RFLP study on 241 E. oleifera accessions, 38 E. guineensis accessions were analyzed using the same 37 cDNA probes. These accessions covered a large part of the geographical distribution areas of these species in America and Africa. In addition, AFLP analysis was performed on a sub-set of 40 accessions of E. oleifera and 22 of E. guineensis using three pairs of enzyme/primer combinations. Data were subjected to Factorial Analysis of Correspondence (FAC) and cluster analysis, with parameters of genetic diversity being also studied. Results appeared congruent between RFLP and AFLP. In the E. oleifera, AFLP confirmed the strong structure of genetic diversity revealed by RFLP, according to geographical origin of the studied material, with the identification of the same four distinct genetic groups: Brazil, French Guyana/Surinam, Peru, north of Colombia/Central America. Both markers revealed that genetic divergence between the two species is of the same magnitude as that among provenances of E. oleifera. This finding is in discrepancy with the supposed early tertiary separation of the two species.

Análise de crescimento de girassol em Latossolo com diferentes níveis de saturação por bases no Cerrado

Com o objetivo de avaliar o comportamento de cinco cultivares de girassol em um Latossolo Vermelho argiloso, foram realizados quatro ensaios sob quatro níveis de saturação por bases (18%, 35%, 56% e 77%) obtidos com aplicação de calcário dolomítico. Os dados semanais de crescimento foram ajustados pelas funções logística, Weibull de três parâmetros e a logística generalizada de quatro parâmetros empregando-se o procedimento NLIN do SAS, bem como os processos interativos do método DUD. A função logística forneceu os melhores ajustes aos dados de crescimento. As cultivares foram categorizadas de acordo com o porte das plantas e tendências de crescimento a partir da análise de agrupamentos (cluster analysis), usando o método das ligações completas e a métrica da distância euclidiana. Todas as cultivares tiveram o crescimento reduzido quando o nível de saturação de bases foi de 18%. A cultivar M734 apresentou bom desempenho no nível de 35% de saturação por bases. Nos demais casos, os melhores resultados foram obtidos nos níveis de saturação por bases entre 56% e 77%. As cultivares EMBRAPA 122 e M738 tiveram redução de crescimento quando a saturação foi elevada para 77%. 'EMBRAPA 122' apresentou maior crescimento inicial e menor altura ao final do ciclo vegetativo.

Consumer acceptance of high pressure processed beef-based chilled ready meals: The mediating role of food-related lifestyle factors

The aim of this research was to investigate the effects of high pressure processing (HPP) on consumer acceptance for chilled ready meals manufactured using a low-value beef cut. Three hundred consumers evaluated chilled ready meals subjected to 4 pressure treatments and a non-treated control monadically on a 9-point scale for liking for beef tenderness and juiciness, overall flavour, overall liking, and purchase intent. Data were also collected on consumers' food consumption patterns, their attitudes towards food by means of the reduced food-related lifestyle (FRL) instrument, and socio-demographics. The results indicated that a pressure treatment of 200 MPa was acceptable to most consumers. K-means cluster analysis identified 4 consumer groups with similar preferences, and the optimal pressure treatments acceptable to specific consumer groups were identified for those firms that would wish to target attitudinally differentiated consumer segments

Characterization of potato genotypes using molecular markers

The objective of this work was to characterize 27 potato genotypes, using molecular markers. Polyacrylamide gel electrophoresis, RAPD techniques and isozymes of esterase, phosphoglucomutase and soluble proteins were analyzed in tubers, and isocitrate dehydrogenase, aspartate transaminase, phosphoglucomutase and peroxidase, in leaves. Eighteen primers were tested and four were chosen, kits OPX (01, 04 and 09) and OPY (07), to analyze RAPD markers in leaf extracts. Similarity and cluster analysis were conducted using Jaccard coefficient and the unweighted pair-group method using arithmetic average. Despite the differences detected in the analysis of proteins and isozymes in the tubers, as well as of isozymes in the leaves, the characterization of all genotypes through gel electrophoresis was not possible, while RAPD markers were efficient to characterize all the 27 genotypes.

Correlation of CXCL10, tumor necrosis factor receptor type II, and galectin 9 with disease activity in juvenile dermatomyositis.

OBJECTIVE: Juvenile dermatomyositis (DM) is a systemic autoimmune disorder of unknown immunopathogenesis in which the immune system targets the microvasculature of skeletal muscles, skin, and other organs. The current mainstay of therapy is a steroid regimen in combination with other immunosuppressive treatments. To date, no validated markers for monitoring disease activity have been identified, which hampers personalized treatment. This study was undertaken to identify a panel of proteins specifically related to active disease in juvenile DM. METHODS: We performed a multiplex immunoassay for plasma levels of 45 proteins related to inflammation in 25 patients with juvenile DM in 4 clinically well-defined groups, as determined by clinical activity and treatment. We compared them to 14 age-matched healthy children and 8 age-matched children with nonautoimmune muscle disease. RESULTS: Cluster analysis of circulating proteins showed distinct profiles for juvenile DM patients and controls based on a group of 10 proteins. In addition to CXCL10, tumor necrosis factor receptor type II (TNFRII) and galectin 9 were significantly increased in active juvenile DM. The levels of these 3 proteins were tightly linked to active disease and correlated with clinical scores (as measured by the Childhood Myositis Assessment Scale and physician's global assessment of disease activity on a visual analog scale). CONCLUSION: Our findings indicate that CXCL10, TNFRII, and galectin 9 correspond to disease status in juvenile DM and thus could be helpful in monitoring disease activity and guiding treatment. Furthermore, they might provide new knowledge about the pathogenesis of this autoimmune disease.

Early-Stage Primary Bone Lymphoma: A Retrospective, Multicenter Rare Cancer Network (RCN) Study.

PURPOSE: Primary bone lymphoma (PBL) represents less than 1% of all malignant lymphomas. In this study, we assessed the disease profile, outcome, and prognostic factors in patients with Stages I and II PBL.¦PATIENTS AND METHODS: Thirteen Rare Cancer Network (RCN) institutions enrolled 116 consecutive patients with PBL treated between 1987 and 2008 in this study. Eighty-seven patients underwent chemoradiotherapy (CXRT) without (78) or with (9) surgery, 15 radiotherapy (RT) without (13) or with (2) surgery, and 14 chemotherapy (CXT) without (9) or with (5) surgery. Median RT dose was 40 Gy (range, 4-60). The median number of CXT cycles was six (range, 2-8). Median follow-up was 41 months (range, 6-242).¦RESULTS: The overall response rate at the end of treatment was 91% (complete response [CR] 74%, partial response [PR] 17%). Local recurrence or progression was observed in 12 (10%) patients and systemic recurrence in 17 (15%). The 5-year overall survival (OS), lymphoma-specific survival (LSS), and local control (LC) were 76%, 78%, and 92%, respectively. In univariate analyses (log-rank test), favorable prognostic factors for OS and LSS were International Prognostic Index (IPI) score ≤1 (p = 0.009), high-grade histology (p = 0.04), CXRT (p = 0.05), CXT (p = 0.0004), CR (p < 0.0001), and RT dose >40 Gy (p = 0.005). For LC, only CR and Stage I were favorable factors. In multivariate analysis, IPI score, RT dose, CR, and CXT were independently influencing the outcome (OS and LSS). CR was the only predicting factor for LC.¦CONCLUSION: This large multicenter retrospective study confirms the good prognosis of early-stage PBL treated with combined CXRT. An adequate dose of RT and complete CXT regime were associated with better outcome.