Ana Rita Vieira

Determination of Beta-Defensin Genomic Copy Number in Different Populations: A Comparison of Three Methods

Background There have been conflicting reports in the literature on association of gene copy number with disease, including CCL3L1 and HIV susceptibility, and β-defensins and Crohns disease. Quantification of precise gene copy numbers is important in order to define any association of gene copy number with disease. At present, real-time quantitative PCR (QPCR) is the most commonly used method to determine gene copy number, however the Paralogue Ratio Test (PRT) is being used in more and more laboratories. Findings In this study we compare a Pyrosequencing-based Paralogue Ratio Test (PPRT) for determining beta-defensin gene copy number with two currently used methods for gene copy number determination, QPCR and triplex PRT by typing five different cohorts (UK, Danish, Portuguese, Ghanaian and Czech) of DNA from a total of 576 healthy individuals. We found a systematic measurement bias between DNA cohorts revealed by QPCR, but not by the PRT-based methods. Using PRT, copy number ranged from 2 to 9 copies, with a modal copy number of 4 in all populations. Conclusions QPCR is very sensitive to quality of the template DNA, generating systematic biases that could produce false-positive or negative disease associations. Both triplex PRT and PPRT do not show this systematic bias, and type copy number within the correct range, although triplex PRT appears to be a more precise and accurate method to type beta-defensin copy number.

NOD2/CARD15 Gene Polymorphisms in Crohn’s Disease: A Genotype-Phenotype Analysis in Danish and Portuguese Patients and Controls

Background: A North-South gradient in Crohn’s disease (CD) implying a higher incidence in northern Europe compared to southern Europe has been established. Aims: To investigate whether there is a difference between Denmark and Portugal in the frequency of CARD15 mutations in CD patients compared to a healthy background population and to compare genotype-phenotype relations in the two countries. Methods: 58 Danish patients and 29 Portuguese patients with CD were matched for age, sex and disease behaviour at time of diagnosis and compared with 200 healthy Danish and Portuguese controls. Phenotypes were recorded at year of diagnosis, 3 years after diagnosis and at end of follow-up. Patients were genotyped for Arg702Trp, Gly908Arg and Leu1007InsC. Results: 22% of the Danish patients vs. 9% of Danish controls compared to 21% of the Portuguese patients vs. 16% had at least one mutation. Mutation rates in Danish patients were significantly different (p = 0.02) compared with Danish controls, no difference (p = 0.51) was found between Portuguese patients and controls. However, a possible relationship between CD and presence of genetic mutations was found when comparing the two countries (p = 0.03) using the Mantel-Haenszel test. No difference in evolution of phenotypes and the CARD15 status in CD was found during follow-up between the two matched populations. Ileal disease correlated to high occurrence of CARD15. Conclusion: No North-South gradient regarding occurrence of CARD15 was revealed. Although a trend towards more mutations in the Portuguese controls was seen, a relationship between CD and CARD15 mutations was observed in both countries.

Discriminating bluemouth, Helicolenus dactylopterus (Pisces: Sebastidae), stocks in Portuguese waters by means of otolith shape analysis

ana neves, vera sequeira, ines farias, ana rita vieira, rafaela paiva and leonel serrano gordo Faculdade de Ciencias da Universidade de Lisboa, Departamento de Biologia Animal & Centro de Oceanografia, Bloco C2, Campo Grande, 1749-016 Lisboa, Portugal, Unidade de Recursos Marinhos e Sustentabilidade, Instituto de Investigacao das Pescas e do Mar (IPIMAR–INRB), Avenida Brasilia, 1449–006 Lisboa, Portugal

Expression profiling of genes involved in the biotrophic colonisation of Coffea arabica leaves by Hemileia vastatrix

Coffee Leaf Rust, caused by Hemileia vastatrix, is the most important disease of Arabica coffee (Coffea arabica), which prompts studies aimed at understanding the genetic basis of this pathogen as well as its complex interaction with the host. In this work, 11 genes, putatively involved in signalling, establishment and maintenance of biotrophy (transport and metabolism), were characterised, and their expression profiles during host infection were assessed by RT-qPCR in three compatible coffee-rust interactions comprising two different rust races. The profiles of two chitin deacetylases (CD) and a heterotrimeric G-protein α subunit transcripts suggest that these enzymes are involved in host-pathogen recognition and establishment of biotrophy at early stages of infection, and the late expression of the CD1 gene was also recorded. Different expression profiles were observed for a MAP kinase gene between the two rust races, suggesting that this gene may be involved in the differentiation of infection structures in a race-specific pattern. Two amino acid transporters, an invertase, a hexose transporter and a mannitol dehydrogenase presented expression profiles similar to those reported in other rust fungi, indicating a fairly conserved genetic programme related to host infection in rust fungi. The strong upregulation of a Uromyces fabae rust transferred protein 1 orthologous gene was observed in H. vastatrix in planta structures, suggesting that this gene may also play a role during the establishment and the maintenance of biotrophy in coffee leaves. Overall, our results provide valuable insights to the current understanding of the biotrophic interaction between H. vastatrix – C. arabica at the molecular level and will contribute to a reasoned and sustainable use of resistant genotypes.

Evolutionary and Biogeographic Insights on the Macaronesian Beta-Patellifolia Species (Amaranthaceae) from a Time-Scaled Molecular Phylogeny.

The Western Mediterranean Region and Macaronesian Islands are one of the top biodiversity hotspots of Europe, containing a significant native genetic diversity of global value among the Crop Wild Relatives (CWR). Sugar beet is the primary crop of the genus Beta (subfamily Betoideae, Amaranthaceae) and despite the great economic importance of this genus, and of the close relative Patellifolia species, a reconstruction of their evolutionary history is still lacking. We analyzed nrDNA (ITS) and cpDNA gene (matK, trnH-psbA, trnL intron, rbcL) sequences to: (i) investigate the phylogenetic relationships within the Betoideae subfamily, and (ii) elucidate the historical biogeography of wild beet species in the Western Mediterranean Region, including the Macaronesian Islands. The results support the Betoideae as a monophyletic group (excluding the Acroglochin genus) and provide a detailed inference of relationships within this subfamily, revealing: (i) a deep genetic differentiation between Beta and Patellifolia species, which may have occurred in Late Oligocene; and (ii) the occurrence of a West-East genetic divergence within Beta, indicating that the Mediterranean species probably differentiated by the end of the Miocene. This was interpreted as a signature of species radiation induced by dramatic habitat changes during the Messinian Salinity Crisis (MSC, 5.96–5.33 Mya). Moreover, colonization events during the Pleistocene also played a role in shaping the current diversity patterns among and within the Macaronesian Islands. The origin and number of these events could not be revealed due to insufficient phylogenetic resolution, suggesting that the diversification was quite recent in these archipelagos, and unravelling potential complex biogeographic patterns with hybridization and gene flow playing an important role. Finally, three evolutionary lineages were identified corresponding to major gene pools of sugar beet wild relatives, which provide useful information for establishing in situ and ex situ conservation priorities in the hotspot area of the Macaronesian Islands.

The gelatinous matrix of the teleost Helicolenus dactylopterus dactylopterus (Delaroche, 1809) in the context of its reproductive strategy

Abstract The bluemouth, Helicolenus dactylopterus dactylopterus (Pisces: Sebastidae), is a zygoparous species with internal fertilization that stores the spermatozoa in crypts within the female ovaries. It spawns gelatinous egg masses that were investigated in this study. Histological analysis confirmed that the ovarian wall and peduncular epithelia play an important role in the production of this gelatinous matrix that contained both oocytes and embryos. Cleavage and blastulae were the most frequent embryonic development stages observed and the tail bud was the latest stage reached. The chemical composition of the matrix was also investigated, revealing that water and proteins are the major components of this substance. This is the first study exclusively dedicated to the investigation of the composition, function and importance of the gelatinous matrix of H. d. dactylopterus in the context of its reproductive strategy.

Development and validation of a visual body condition scoring system for dairy goats with picture-based training

Body condition scoring (BCS) is the most widely used method to assess changes in body fat reserves, which reflects its high potential to be included in on-farm welfare assessment protocols. Currently used scoring systems in dairy goats require animal restraint for body palpation. In this study, the Animal Welfare Indicators project (AWIN) proposes to overcome this constraint by developing a scoring system based only on visual assessment. The AWIN visual body condition scoring system highlights representative animals from 3 categories: very thin, normal, and very fat, and was built from data sets with photographs of animals scored by a commonly used 6-point scoring system that requires palpation in 2 anatomical regions. Development of the AWIN scoring system required 3 steps: (1) identification and validation of a body region of interest; (2) sketching the region from photographs; and (3) creation of training material. The scoring systems reliability was statistically confirmed. An initial study identified features in the rump region from which we could compute a set of body measurements (i.e., measures based on anatomical references of the rump region) that showed a strong correlation with the assigned BCS. To validate the result, we collected a final data set from 171 goats. To account for variability in animal size and camera position, we mapped a subset of features to a standard template and aligned all the rump images before computing the body measurements. Scientific illustrations were created from the aligned images of animals identified as representative of each category to increase clarity and reproducibility. For training material, we created sketches representing the threshold between consecutive categories. Finally, we conducted 2 field reliability studies. In the first test, no training was given to 4 observers, whereas in the second, training using the threshold images was delivered to the same observers. In the first experiment, interobserver results was substantial, showing that the visual scoring system is clear and unambiguous. Moreover, results improved after training, reaching almost perfect agreement for the very fat category. The visual body condition scoring system is not only a practical tool for BCS in dairy goats but also shows potential to be fully automated, which would enhance its use in welfare assessment schemes and farm management.

Results of testing the prototype of the AWIN welfare assessment protocol for dairy goats in 30 intensive farms in Northern Italy

Abstract The AWIN project aimed at developing an on-farm welfare assessment protocol for adult dairy goats. A prototype protocol was tested in 30 intensive dairy goat farms to evaluate its feasibility in farms of different size. Time for applying the prototype was recorded and any other constraint was taken into account. Moreover, data collected during the prototype testing provided information on the prevalence of welfare issues in intensive dairy goat farms in Northern Italy. The prototype included 25 animal-based indicators (14 group- and 11 individual-level indicators). The prototype showed a good on-farm feasibility and it was highly accepted among stakeholders, as its application did not interfere with the daily routine. Approximately 2 h were required for the application of the prototype. When feeding racks were available, using them for locking the animals during the individual assessment resulted advantageous to speed the data collection and to reduce handling stress to the goats and disturbance to the farmers. Farm size and different management systems influenced the prevalence of some indicators, with small farms in general better welfare conditions compared to larger farms. The results of the present study represent an important starting point to set up an epidemiological database that may lead to improve the welfare status of goats.

B-cell phenotype and IgD-CD27- memory B cells are affected by TNF-inhibitors and tocilizumab treatment in rheumatoid arthritis.

Background The use of TNF-inhibitors and/or the IL-6 receptor antagonist, tocilizumab, in rheumatoid arthritis (RA) have pleiotropic effects that also involve circulating B-cells. The main goal of this study was to assess the effect of TNF-inhibitors and tocilizumab on B-cell phenotype and gene expression in RA. Methods Blood samples were collected from untreated early RA (ERA) patients, established RA patients under methotrexate treatment, established RA patients before and after treatment with TNF-inhibitors and tocilizumab, and healthy donors. B-cell subpopulations were characterized by flow cytometry and B-cell gene expression was analyzed by real-time PCR on isolated B-cells. Serum levels of BAFF, CXCL13 and sCD23 were determined by ELISA. Results The frequency of total CD19+ B cells in circulation was similar between controls and all RA groups, irrespective of treatment, but double negative (DN) IgD-CD27- memory B cells were significantly increased in ERA and established RA when compared to controls. Treatment with TNF-inhibitors and tocilizumab restored the frequency of IgD-CD27- B-cells to normal levels, but did not affect other B cell subpopulations. TACI, CD95, CD5, HLA-DR and TLR9 expression on B-cells significantly increased after treatment with either TNF-inhibitors and/ or tocilizumab, but no significant changes were observed in BAFF-R, BCMA, CD69, CD86, CXCR5, CD23, CD38 and IgM expression on B-cells when comparing baseline with post-treatment follow-ups. Alterations in B-cell gene expression of BAFF-R, TACI, TLR9, FcγRIIB, BCL-2, BLIMP-1 and β2M were found in ERA and established RA patients, but no significant differences were observed after TNF-inhibitors and tocilizumab treatment when comparing baseline and follow-ups. Serum levels of CXCL13, sCD23 and BAFF were not significantly affected by treatment with TNF-inhibitors and tocilizumab. Conclusions In RA patients, the use of TNF-inhibitors and/ or tocilizumab treatment affects B-cell phenotype and IgD-CD27- memory B cells in circulation, but not B-cell gene expression levels.

Effect of greenhouse conditions on the leaf apoplastic proteome of Coffea arabica plants

UNLABELLED This work describes the coffee leaf apoplastic proteome and its modulation by the greenhouse conditions. The apoplastic fluid (APF) was obtained by leaf vacuum infiltration, and the recovered proteins were separated by 2-DE and subsequently identified by matrix assisted laser desorption/ionization time of flight-mass spectrometry, followed by homology search in EST coffee databases. Prediction tools revealed that the majority of the 195 identified proteins are involved in cell wall metabolism and in stress/defense responses. Although most of the proteins follow the classical secretory mechanism, a low percentage of them seem to result from unconventional secretion (leaderless secreted proteins). Principal components analysis revealed that the APF samples formed two distinct groups, with the temperature amplitude mostly contributing for this separation (higher or lower than 10°C, respectively). Sixty one polypeptide spots allowed defining these two groups and 28 proteins were identified, belonging to carbohydrate metabolism, cell wall modification and proteolysis. Interestingly stress/defense proteins appeared as more abundant in Group I which is associated with a higher temperature amplitude. It seems that the proteins in the coffee leaf APF might be implicated in structural modifications in the extracellular space that are crucial for plant development/adaptation to the conditions of the prevailing environment. BIOLOGICAL SIGNIFICANCE This is the first detailed proteomic study of the coffee leaf apoplastic fluid (APF) and of its modulation by the greenhouse conditions. The comprehensive overview of the most abundant proteins present in the extra-cellular compartment is particularly important for the understanding of coffee responses to abiotic/biotic stress. This article is part of a Special Issue entitled: Environmental and structural proteomics.