Structural analysis and involvement in plant innate immunity of Xanthomonas axonopodis pv. citri lipopolysaccharide

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker, provoking defoliation and premature fruit drop with concomitant economical damage. In plant pathogenic bacteria, lipopolysaccharides are important virulence factors, and they are being increasingly recognized as major pathogen-associated molecular patterns for plants. In general, three domains are recognized in a lipopolysaccharide: the hydrophobic lipid A, the hydrophilic O-antigen polysaccharide, and the core oligosaccharide, connecting lipid A and O-antigen. In this work, we have determined the structure of purified lipopolysaccharides obtained from Xanthomonas axonopodis pv. citri wild type and a mutant of the O-antigen ABC transporter encoded by the wzt gene. High pH anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrum analysis were performed, enabling determination of the structure not only of the released oligosaccharides and lipid A moieties but also the intact lipopolysaccharides. The results demonstrate that Xac wild type and Xacwzt LPSs are composed mainly of a penta- or tetra-acylated diglucosamine backbone attached to either two pyrophosphorylethanolamine groups or to one pyrophosphorylethanolamine group and one phosphorylethanolamine group. The core region consists of a branched oligosaccharide formed by Kdo₂Hex₆GalA₃Fuc3NAcRha₄ and two phosphate groups. As expected, the presence of a rhamnose homo-oligosaccharide as O-antigen was determined only in the Xac wild type lipopolysaccharide. In addition, we have examined how lipopolysaccharides from Xac function in the pathogenesis process. We analyzed the response of the different lipopolysaccharides during the stomata aperture closure cycle, the callose deposition, the expression of defense-related genes, and reactive oxygen species production in citrus leaves, suggesting a functional role of the O-antigen from Xac lipopolysaccharides in the basal response.     

Use of planar kV vs. CBCT in evaluation of setup errors in oesophagus carcinoma radiotherapy

Aim

The aim of this study is to evaluate differences in terms of the setup errors observed using kV planar image compared to CBCT for oesophageal cancer patients.

Background

Planar kV images are quick to acquire but only allow the observation of bony structures. CBCT allows the evaluation of soft tissues, which includes the oesophagus (and tumour) and OAR, giving a more accurate verification of the positioning.

Materials and Methods

All patients were imaged with both techniques between January 2012 and March 2014 were included in the study (16 patients, 212 kV images and 116 CBCT images). Differences between the setup errors observed on the two images modalities were studied. A correlation study between TNM staging, tumour location and immobilization systems with setup errors was also done. Finally, the calculation of systematic and random errors allowed to determine the CTV–PTV margin.

Results

A significant discrepancy (p < 0.05) between the setup errors observed with kV and CBCT was observed in the lateral direction. No statistical correlation was found between setup errors and tumour location, immobilization system or TNM staging. The CTV–PTV margin was smaller with CBCT in the vertical (0.6 cm vs. 0.9 cm) and longitudinal (0.7 cm vs. 1 cm) directions and smaller with kV for the lateral directions (0.8 cm vs. 0.9 cm).

Conclusions

The chosen modality influences the setup error observed which will influence the correction applied. Allowing a better observation of the volumes of interest, CBCT should be the modality of choice in this pathology. The CTV–PTV margins could be shrunk if CBCT is used.     

Effect of water temperature and dietary starch on growth and metabolic utilization of diets in gilthead sea bream (Sparus aurata) juveniles

We evaluated the effect of dietary starch level on growth performance, feed utilization, whole-body composition and activity of selected key enzymes of intermediary metabolism in gilthead sea bream juveniles reared at 18 and 25 degrees C. A diet was formulated to contain 48% crude protein, 12% lipids and 30% gelatinized maize starch (diet 30GS). Two other diets were formulated to include the same level of ingredients as diet 30GS except for the gelatinized starch, which was included at 20% (diet 20GS) or 10% (diet 10GS). No adjustment to diet composition was otherwise made. Each diet was fed to triplicate groups of gilthead sea bream (30 g initial mass) for 8 weeks, on a pair-feeding scheme. The higher temperature improved growth performance but the opposite was true for feed efficiency and protein efficiency ratio. Independently of temperature, growth performance, feed efficiency and protein efficiency ratio were lower in fish fed diet 30GS. No effect of temperature or dietary starch level on whole-body composition was noticed. Hepatosomatic index and liver glycogen were higher at 18 degrees C and, within each temperature, in fish fed diet 30GS. Glycemia was not affected by temperature, but was lower in fish fed diet 10GS. Data on enzyme activities showed that increasing water temperature enhances liver glucokinase (GK) and pyruvate kinase (PK) activities, suggesting that gilthead sea bream is more apt to use dietary starch at higher temperatures. No effect of temperature was noticed on hexokinase (HK), fructose-1,6-bisphosphatase (FBPase), glucose-6-phosphate dehydrogenase (G6PD) and glutamate dehydrogenase (GDH) activities. Dietary starch enhanced PK and FBPase activities while depressed GDH activity, suggesting a lack of significant regulation of hepatic glucose utilization and production in this species. HK, GK and G6PD activities were unaffected by dietary composition. Irrespectively of water temperature, gelatinized starch may be included up to 20% in diets for gilthead sea bream juveniles; at higher dietary levels, growth and efficiency of feed utilization are depressed.     

Amelioration of ligninolytic enzyme production by Phanerochaete chrysosporium in airlift bioreactors

Maximum activities of manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) in free cultures of Phanerochaete chrysosporium (ATCC 24725) were 258 U l^–1 and 103 U l^–1, respectively, in an airlift bioreactor. Immobilisation of the fungus on an inert carrier as well as several design modifications of the bioreactor employed gave MnP activities around 500–600 U l^–1 during 9 days' operation. The continuous operation of the latter led to MnP and LiP activities about 140 U l^–1 and 100 U l^–1, respectively, for two months, without operational problems. Furthermore, the extracellular liquid secreted decolourised the polymeric dye Poly R-478 about 56%.     

Fatal haemorrhage and neoplastic thrombosis in a captive African lion (<Emphasis Type="Italic">Panthera leo</Emphasis>) with metastatic testicular sex cord–stromal tumour

Background

The study of neoplasia in wildlife species contributes to the understanding of cancer biology, management practices, and comparative pathology. Higher frequencies of neoplasms among captive non-domestic felids have been reported most commonly in aging individuals. However, testicular tumours have rarely been reported. This report describes a metastatic testicular sex cord–stromal tumour leading to fatal haemorrhage and thrombosis in a captive African lion (Panthera leo).

Case presentation

During necropsy of a 16-year-old male African lion, the left testicle and spermatic cord were found to be intra-abdominal (cryptorchid), semi-hard and grossly enlarged with multiple pale-yellow masses. Encapsulated haemorrhage was present in the retroperitoneum around the kidneys. Neoplastic thrombosis was found at the renal veins opening into the caudal vena cava. Metastases were observed in the lungs and mediastinal lymph nodes. Histology revealed a poorly differentiated pleomorphic neoplasm comprised of round to polygonal cells and scattered spindle cells with eosinophilic cytoplasm. An immunohistochemistry panel of inhibin-α, Ki-67, human placental alkaline phosphatase, cytokeratin AE1/AE3, cKit, vimentin and S100 was conducted. Positive cytoplasmic immunolabeling was obtained for vimentin and S100.

Conclusions

The gross, microscopic and immunohistochemical findings of the neoplasm were compatible with a poorly differentiated pleomorphic sex cord–stromal tumour. Cause of death was hypovolemic shock from extensive retroperitoneal haemorrhage and neoplastic thrombosis may have contributed to the fatal outcome. To our knowledge, this is the first report of sex cord–stromal tumour in non-domestic felids.

     

Distribution of the related weevil species Sitophilus oryzae and S. zeamais in Brazil

The genus Sitophilus （Coleoptera： Curculionidae） encompasses species of great economic importance as stored grain pests worldwide. Among these species, the maize and the rice weevils （Sitophilus zeamais and Sitophilus oryzae, respectively） are partic- ularly important in warmer climates. These two weevils exhibit closely morphological and ecological resemblance making difficult their proper identification and recognition of their distribution in grain-producing regions. Both species are recorded in South America and particularly in Brazil, but their respective distribution and prevalence were not yet assessed in the region. Therefore, several insect samples throughout Brazil were collected and subjected to morphological identification using male genitalia and also using molec- ular identification with species-specific primers designed for clear recognition of both the species. The primers were designed for the specific amplification of a gene fragment of the cytochrome oxidase subunit I, which exhibited high specificity during our prelimi- nary experiments with insects from six populations of known species （either S. zeamais or S. oryzae）. Both identification strategies provided the same results indicating preva- lence of the maize weevil S. zeamais throughout the country. Two hypotheses may explain such prevalence： （i） the likely host preference ofS. zeamais for maize because this is the most cultivated cereal in Brazil, and （ii） the prevalence ofS. zeamais in tropical regions as compared with S. oryzae, which is more disseminated in subtropical and temperate regions.     

Rescue of F508del-CFTR by RXR motif inactivation triggers proteome modulation associated with the unfolded protein response

F508del-CFTR, the most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, disrupts intracellular trafficking leading to cystic fibrosis (CF). The trafficking defect of F508del-CFTR can be rescued by simultaneous inactivation of its four RXR motifs (4RK). Proteins involved in the F508del-CFTR trafficking defect and/or rescue are therefore potential CF therapeutic targets. We sought to identify these proteins by investigating differential proteome modulation in BHK cells over-expressing wt-CFTR, F508del-CFTR or the revertant F508del/4RK-CFTR. By 2-dimensional electrophoresis-based proteomics and western blot approaches we demonstrated that over-expression of F508del/4RK-CFTR modulates the expression of a large number of proteins, many of which are reported interactors of CFTR and/or 14-3-3 with potential roles in CFTR trafficking. GRP78/BiP, a marker of ER stress and unfolded protein response (UPR), is up-regulated in cells over-expressing either F508del-CFTR or F598del/4RK-CFTR. However, over-expression of F508del/4RK-CFTR induces the up-regulation of many other UPR-associated proteins (e.g. GRP94, PDI, GRP75/mortalin) and, interestingly, the down-regulation of proteasome components associated with CFTR degradation, such as the proteasome activator PA28 (PSME2) and COP9 signalosome (COPS5/CSN5). Moreover, the F508del-CFTR-induced proteostasis imbalance, which involves some heat shock chaperones (e.g. HSP72/Hpa2), ER-EF-hand Ca²⁺-binding proteins (calumenin) and the proteasome activator PA28 (PSME2), tends to be ‘restored’, i.e., in BHK cells over-expressing F508del/4RK-CFTR those proteins tend to have expression levels similar to the wild-type ones. These findings indicate that a particular cellular environment orchestrated by the UPR contributes to and/or is compatible with F508del/4RK-CFTR rescue.     

Environmental favorability models as a tool to support trophic rewilding initiatives

Trophic rewilding is considered a conservation measure that aims to restore the trophic interactions. In north-central Portugal, the Iberian wolf (Canis lupus signatus) is endangered mainly due to human persecution, triggered by wolf depredation on livestock. Several initiatives have occurred in this area to increase wolf wild prey availability by reintroducing/reinforcing roe deer (Capreolus capreolus) populations. However, the success of trophic rewilding initiatives relies on up-to-date scientific evidence, considering both ecological and social settings. Favorability models can be used to infer the most suitable habitats for roe deer settlement and expansion by analyzing the environmental variables known to influence species ecology and behavior. In this study, we infer how the prey habitat favorability can contribute to plan prey populations reinforcement processes as a tool for trophic chain restoration. Using 330 camera traps, we monitored the roe deer population over 8,940 km² in north-central Portugal. Field data were used to evaluate the environmental determinants of roe deer presence. By using the favorability function, we created a spatially explicit gradient of habitat favorability that allowed us to define key areas for the application of management measures. The selected sites are strategically positioned to promote the natural expansion of established roe deer populations and overlap the current distribution of Iberian wolf. These results are an important step toward the restoration of the trophic chain and to reduce human–wolf conflicts. Our proposal paves the way for future rewilding initiatives, especially where prey species are central to the survival of top predators.