Evaluation of PCR Based on Gene <Emphasis Type="Italic">apxIVA</Emphasis> Associated with 16S rDNA Sequencing for the Identification of <Emphasis Type="Italic">Actinobacillus pleuropneumoniae</Emphasis> and Related Species

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.     

Biphasic regulation of HMG-CoA reductase expression and activity during wound healing and its functional role in the control of keratinocyte angiogenic and proliferative responses

In this study, we determined the regulation and potential function of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (HMGR) during skin repair in mice. Upon skin injury, healthy mice exhibited a biphasic increase in HMGR expression and activity with elevated levels at days 3 and 13 post-wounding. In situ hybridization revealed wound margin keratinocytes as a cellular source of HMGR expression. In vitro experiments using cultured HaCaT keratinocytes uncovered epidermal growth factor (EGF), transforming growth factor (TGF)-alpha, and insulin as potent co-inducers of HMGR activity and vascular endothelial growth factor (VEGF) in the cells. Insulin-, but not EGF-mediated VEGF protein expression was functionally connected to co-induced HMGR activity, as simvastatin restrictively interfered only with insulin-induced translation of VEGF mRNA by inhibition of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) phosphorylation. Functional ablation of insulin-induced sterol regulatory element-binding protein (SREBP)-2 by siRNA abolished HMGR expression and insulin-triggered VEGF protein release from keratinocytes. Simvastatin also blocked proliferation of cultured keratinocytes. The observed inhibitory effects of simvastatin on keratinocyte VEGF expression and proliferation could be reversed by mevalonate, the product of HMGR enzymatic activity. In accordance, simvastatin-mediated inhibition of HMGR activity in acutely regenerating tissue of wounded mice was paralleled by a marked loss of VEGF protein expression and disturbances of normal proliferation processes in wound margin keratinocytes during skin repair.     

Modelling the distribution and compositional variation of plant communities at the continental scale

Aim

We investigate whether (1) environmental predictors allow to delineate the distribution of discrete community types at the continental scale and (2) how data completeness influences model generalization in relation to the compositional variation of the modelled entities.

Location

Europe.

Methods

We used comprehensive datasets of two community types of conservation concern in Europe: acidophilous beech forests and base‐rich fens. We computed community distribution models (CDMs) calibrated with environmental predictors to predict the occurrence of both community types, evaluating geographical transferability, interpolation and extrapolation under different scenarios of sampling bias. We used generalized dissimilarity modelling (GDM) to assess the role of geographical and environmental drivers in compositional variation within the predicted distributions.

Results

For the two community types, CDMs computed for the whole study area provided good performance when evaluated by random cross‐validation and external validation. Geographical transferability provided lower but relatively good performance, while model extrapolation performed poorly when compared with interpolation. Generalized dissimilarity modelling showed a predominant effect of geographical distance on compositional variation, complemented with the environmental predictors that also influenced habitat suitability.

Main conclusions

Correlative approaches typically used for modelling the distribution of individual species are also useful for delineating the potential area of occupancy of community types at the continental scale, when using consistent definitions of the modelled entity and high data completeness. The combination of CDMs with GDM further improves the understanding of diversity patterns of plant communities, providing spatially explicit information for mapping vegetation diversity and related habitat types at large scales.

     

alpha1beta1 Integrin+ and regulatory Foxp3+ T cells constitute two functionally distinct human CD4+ T cell subsets oppositely modulated by TNFalpha blockade

The expression of the collagen receptor alpha(1)beta(1) integrin (VLA-1) on CD4(+) T cells is largely restricted to CCR7(-)CD45RO(+) cells that localize to inflamed tissues. Moreover, neutralizing alpha(1) integrin, in vivo, has been shown to compromise cell-mediated immunity. Our current study shows that the expression of VLA-1 on human CD4(+) T cells is restricted to conventional effectors. In contrast, Foxp3(+) T regulatory cells (Tregs) do not express this receptor. Moreover, Foxp3 or VLA-1 expression remained a mutually exclusive event in CD4(+) T cells even upon polyclonal anti-CD3-induced activation. Because TNFalpha blockade ameliorates certain T cell-dependent autoimmune disorders in humans, we investigated, in vitro, whether neutralizing TNFalpha affected the balance between the proinflammatory VLA-1(+) effectors and the counteracting Tregs. We found that anti-CD3 stimulation of freshly isolated PBL from healthy individuals, coupled with continuous TNFalpha blockade, inhibited the typical activation-dependent generation of CD4(+)VLA-1(+) Th1 cells. In contrast, it augmented the outgrowth of VLA-1(neg/dim)CD25(high) and Foxp3(+)CD4(+) T cells. Indeed, repeated anti-CD3 stimulation coupled with TNFalpha blockade generated CD4(+) T cell lines enriched for VLA-1(-)Foxp3(+) Tregs. Importantly, these CD4(+) T cells displayed potent suppressive functions toward autologous CD4(+) PBL, including the suppression of the activation-dependent induction of VLA-1(+) effectors. Thus, we propose a novel mechanism by which anti-TNFalpha therapy may restore self-tolerance, by shifting the balance between VLA-1(+) effectors and Foxp3(+) Tregs, during immune activation, in favor of the latter suppressor cell population.     

Comparative response of Metarhizium brunneum to the cuticles of susceptible and resistant hosts

Earlier studies demonstrated that Metarhizium brunneum, usually a broad-host pathogen of arthropods, is unable to complete its pathogenic life cycle when inoculated on the fungus-resistant tick, Hyalomma excavatum engorged females. While the fungus penetrates the cuticle of fungus-susceptible tick, Rhipicephalus annulatus females, it is unable to penetrate the cuticle of fungus-resistant tick, and even perishes on its surface. This is probably due to high concentration of antifungal fatty acids and probably also due to a hypersensitive-like response of the tick. To understand the metabolic pathways occurring in the fungal hyphae upon encountering the cuticles, we compared the response of the fungus to cuticle from susceptible and resistant tick cuticles by 2D-gels. The intracellular proteomes of M. brunneum Mb7 exposed to cuticle of the fungus-susceptible tick, R. annulatus, and to the fungus-resistant tick, H. excavatum engorged females were compared after exposure to either cuticles. By means of liquid chromatography-mass spectrometry/mass spectrometry we identified in both proteomes common proteins involved in biological processes as well as unique proteins identified only in the proteome of fungus exposed to fungus-resistant tick cuticle. These proteins were identified in high probability as heat shock proteins, four key enzymes of the glyoxylate cycle, and proteins associated with hypoxia, and exposure to antifungal drugs. These findings are discussed within the M. brunneum-tick pathosystem in relation to tick resistance and host resistance in general.     

Proteins modified with DNAzymes or aptamers act as biosensors or biosensor labels

Hybrid systems composed of a glucose oxidase (GOx)/peroxidase-mimicking DNAzyme, and of microperoxidase-11 (MP-11)/anti-thrombin aptamer were synthesized. The hybrid systems were employed as amplifying labels for the colorimetric or chemiluminescence detection of an enzyme functions, and thrombin analysis, respectively. In the GOx/DNAzyme system, the GOx-mediated oxidation of glucose led to the formation of H(2)O(2), and this activated the oxidation of ABTS to a colored product, or to the generation of chemiluminescence in the presence of luminol. The MP-11/anti-thrombin aptamer enabled the amplified analysis of thrombin by the MP-11-mediated generation of chemiluminescence in the presence of luminol/H(2)O(2).     

Isolation and characterization of cellulolytic bacteria from the Stain house Lake,Antarctica

The main aim was to evaluate the occurrence of cellulolytic bacteria from the Stain house Lake, located at Admiralty Bay, Antarctica. Thick cotton string served as a cellulose bait for the isolation of bacteria. A total of 52 bacterial isolates were recovered and tested for their cellulase activity, and two of them, isolates CMAA 1184 and CMAA 1185, showed significant cellulolytic activity on carboxymethylcellulose agar plates. Phylogenetic analysis placed the isolates into the Bacillus 16S ribosomal RNA gene subclade. Both isolates produced a cold-active cellulase which may play a crucial role in this extreme environment.     

Oxidation of an Exposed Methionine Instigates the Aggregation of Glyceraldehyde-3-phosphate Dehydrogenase

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a ubiquitous and abundant protein that participates in cellular energy production. GAPDH normally exists in a soluble form; however, following necrosis, GAPDH and numerous other intracellular proteins convert into an insoluble disulfide-cross-linked state via the process of “nucleocytoplasmic coagulation.” Here, free radical-induced aggregation of GAPDH was studied as an in vitro model of nucleocytoplasmic coagulation. Despite the fact that disulfide cross-linking is a prominent feature of GAPDH aggregation, our data show that it is not a primary rate-determining step. To identify the true instigating event of GAPDH misfolding, we mapped the post-translational modifications that arise during its aggregation. Solvent accessibility and energy calculations of the mapped modifications within the context of the high resolution native GAPDH structure suggested that oxidation of methionine 46 may instigate aggregation. We confirmed this by mutating methionine 46 to leucine, which rendered GAPDH highly resistant to free radical-induced aggregation. Molecular dynamics simulations suggest that oxidation of methionine 46 triggers a local increase in the conformational plasticity of GAPDH that likely promotes further oxidation and eventual aggregation. Hence, methionine 46 represents a “linchpin” whereby its oxidation is a primary event permissive for the subsequent misfolding, aggregation, and disulfide cross-linking of GAPDH. A critical role for linchpin residues in nucleocytoplasmic coagulation and other forms of free radical-induced protein misfolding should now be investigated. Furthermore, because disulfide-cross-linked aggregates of GAPDH arise in many disorders and because methionine 46 is irrelevant to native GAPDH function, mutation of methionine 46 in models of disease should allow the unequivocal assessment of whether GAPDH aggregation influences disease progression.     

Brazilian Microbiome Project: Revealing the Unexplored Microbial Diversity—Challenges and Prospects

The Brazilian Microbiome Project (BMP) aims to assemble a Brazilian Metagenomic Consortium/Database. At present, many metagenomic projects underway in Brazil are widely known. Our goal in this initiative is to co-ordinate and standardize these together with new projects to come. It is estimated that Brazil hosts approximately 20 % of the entire world’s macroorganism biological diversity. It is 1 of the 17 countries that share nearly 70 % of the world’s catalogued animal and plant species, and is recognized as one of the most megadiverse countries. At the end of 2012, Brazil has joined GBIF (Global Biodiversity Information Facility), as associated member, to improve the access to the Brazilian biodiversity data in a free and open way. This was an important step toward increasing international collaboration and clearly shows the commitment of the Brazilian government in directing national policies toward sustainable development. Despite its importance, the Brazilian microbial diversity is still considered to be largely unknown, and it is clear that to maintain ecosystem dynamics and to sustainably manage land use, it is crucial to understand the biological and functional diversity of the system. This is the first attempt to collect and collate information about Brazilian microbial genetic and functional diversity in a systematic and holistic manner. The success of the BMP depends on a massive collaborative effort of both the Brazilian and international scientific communities, and therefore, we invite all colleagues to participate in this project.