Toll receptors type-2 and CR3 expression of canine monocytes and its correlation with immunohistochemistry and xenodiagnosis in visceral leishmaniasis

The aim of the present study was to investigate TLR2 expression in peripheral blood monocytes from dogs naturally infected with Leishmania (Leishmania) infantum to determine whether it correlates with CD11b/CD18 (CR3) expression, and to evaluate the potential of dogs as sources of infection using phlebotomine xenodiagnosis. Forty eight dogs were serologically diagnosed with L. infantum infection by indirect immunofluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA). Parasitological exams from bone-marrow aspirates were positive by PCR analysis. All dogs were clinical defined as symptomatic. Ear skin tissue samples were obtained for immunohistochemistry (IHQ) analysis. The potential of these dogs as a source of infection using phlebotomine xenodiagnosis (XENO) was evaluated. Flow cytometry was carried out on peripheral blood mononuclear cells using superficial receptors including CD14, CD11b, TLR2 and MHCII. IHQ ear skin tissue parasite load and XENO where done where we found a strict correlation (r = 0.5373). Dogs with higher expression of MFI of CD11b inside CD14 monocytes were represented by dogs without parasite ear tissue load that were unable to infect phlebotomines (IHQ⁻/XENO⁻). Dogs with lower expression of MFI of CD11b inside CD14 monocytes were represented by dogs with parasite ear tissue load and able to infect phlebotomines (IHQ⁺/XENO⁺) (p = 0,0032). Comparable results were obtained for MFI of MHCII (p = 0.0054). In addition, considering the population frequency of CD11b⁺TLR2⁺ and CD11b⁺MHCII⁺, higher values were obtained from dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺ (p = 0.01; p = 0.0048, respectively). These data, together with the TLR2 and NO assays results (CD11b⁺TLR2⁺ and NO with higher values for dogs with IHQ⁻/XENO⁻ than dogs with IHQ⁺/XENO⁺), led to the conclusion that IHQ⁻/XENO⁻ dogs are more resistant or could modulate the cellular immune response essential for Leishmania tissue clearance.     

CPORT: a consensus interface predictor and its performance in prediction-driven docking with HADDOCK

Background

Macromolecular complexes are the molecular machines of the cell. Knowledge at the atomic level is essential to understand and influence their function. However, their number is huge and a significant fraction is extremely difficult to study using classical structural methods such as NMR and X-ray crystallography. Therefore, the importance of large-scale computational approaches in structural biology is evident. This study combines two of these computational approaches, interface prediction and docking, to obtain atomic-level structures of protein-protein complexes, starting from their unbound components.

Methodology/Principal Findings

Here we combine six interface prediction web servers into a consensus method called CPORT (Consensus Prediction Of interface Residues in Transient complexes). We show that CPORT gives more stable and reliable predictions than each of the individual predictors on its own. A protocol was developed to integrate CPORT predictions into our data-driven docking program HADDOCK. For cases where experimental information is limited, this prediction-driven docking protocol presents an alternative to ab initio docking, the docking of complexes without the use of any information. Prediction-driven docking was performed on a large and diverse set of protein-protein complexes in a blind manner. Our results indicate that the performance of the HADDOCK-CPORT combination is competitive with ZDOCK-ZRANK, a state-of-the-art ab initio docking/scoring combination. Finally, the original interface predictions could be further improved by interface post-prediction (contact analysis of the docking solutions).

Conclusions/Significance

The current study shows that blind, prediction-driven docking using CPORT and HADDOCK is competitive with ab initio docking methods. This is encouraging since prediction-driven docking represents the absolute bottom line for data-driven docking: any additional biological knowledge will greatly improve the results obtained by prediction-driven docking alone. Finally, the fact that original interface predictions could be further improved by interface post-prediction suggests that prediction-driven docking has not yet been pushed to the limit. A web server for CPORT is freely available at http://haddock.chem.uu.nl/services/CPORT.     

Global conservation priorities for marine turtles

Where conservation resources are limited and conservation targets are diverse, robust yet flexible priority-setting frameworks are vital. Priority-setting is especially important for geographically widespread species with distinct populations subject to multiple threats that operate on different spatial and temporal scales. Marine turtles are widely distributed and exhibit intra-specific variations in population sizes and trends, as well as reproduction and morphology. However, current global extinction risk assessment frameworks do not assess conservation status of spatially and biologically distinct marine turtle Regional Management Units (RMUs), and thus do not capture variations in population trends, impacts of threats, or necessary conservation actions across individual populations. To address this issue, we developed a new assessment framework that allowed us to evaluate, compare and organize marine turtle RMUs according to status and threats criteria. Because conservation priorities can vary widely (i.e. from avoiding imminent extinction to maintaining long-term monitoring efforts) we developed a “conservation priorities portfolio” system using categories of paired risk and threats scores for all RMUs (n = 58). We performed these assessments and rankings globally, by species, by ocean basin, and by recognized geopolitical bodies to identify patterns in risk, threats, and data gaps at different scales. This process resulted in characterization of risk and threats to all marine turtle RMUs, including identification of the world''s 11 most endangered marine turtle RMUs based on highest risk and threats scores. This system also highlighted important gaps in available information that is crucial for accurate conservation assessments. Overall, this priority-setting framework can provide guidance for research and conservation priorities at multiple relevant scales, and should serve as a model for conservation status assessments and priority-setting for widespread, long-lived taxa.     

COMMD1-mediated ubiquitination regulates CFTR trafficking

The CFTR (cystic fibrosis transmembrane conductance regulator) protein is a large polytopic protein whose biogenesis is inefficient. To better understand the regulation of CFTR processing and trafficking, we conducted a genetic screen that identified COMMD1 as a new CFTR partner. COMMD1 is a protein associated with multiple cellular pathways, including the regulation of hepatic copper excretion, sodium uptake through interaction with ENaC (epithelial sodium channel) and NF-kappaB signaling. In this study, we show that COMMD1 interacts with CFTR in cells expressing both proteins endogenously. This interaction promotes CFTR cell surface expression as assessed by biotinylation experiments in heterologously expressing cells through regulation of CFTR ubiquitination. In summary, our data demonstrate that CFTR is protected from ubiquitination by COMMD1, which sustains CFTR expression at the plasma membrane. Thus, increasing COMMD1 expression may provide an approach to simultaneously inhibit ENaC absorption and enhance CFTR trafficking, two major issues in cystic fibrosis.     

Systematics and phylogeny of Potamotrygonocestus (Platyhelminthes, Tetraphyllidea, Onchobothriidae) with descriptions of three new species from freshwater potamotrygonids (Myliobatoidei, Potamotrygonidae)

Specimens of Potamotrygonocestus used in the present taxonomic and phylogenetic study were collected in freshwater potamotrygonids from the Rio Paraná (Puerto Reconquista, Santa Fé, Argentina; 59°42'W, 29°29'S) during February 1995, and from the Amazon region of Brazil during September to December 1996, supplemented by specimens previously deposited in museum collections. Six species of Potamotrygonocestus were recognized ( P . amazonensis , P . chaoi sp. n., P . fitzgeraldae sp. n., P . magdalenensis , P . maurae sp. n., P . travassosi ), for which an identification key is provided. Potamotrygonocestus orinocoensis is considered a junior synonym of P . travassosi . Additional specimens of Potamotrygonocestus may represent two undescribed species. A phylogenetic analysis of six morphological characters for the six named species of Potamotrygonocestus generated a single tree eight steps long, with a consistency index of 0.87, a retention index of 0.90, and a rescaled consistency index of 0.79 with the following topology: ( P . maurae sp. n. (( P . amazonensis  +  P . magdalenensis ) ( P . chaoi sp. n. ( P . fitzgeraldae sp. n. +  P . travassosi )))).     

Purification of Human Recombinant GATA-1 from Bacteria: Implication for Protein-Protein Interaction Studies

GATA-1 is a key regulator of terminal erythroid differentiation in mammals and birds. The structural and biochemical studies of human GATA-1 (hGATA-1) are limited by the difficulty of its purification in a sufficient amount. Here we describe the procedure for obtaining pure bacterial recombinant hGATA-1 in an active functional state. We demonstrate that this protein may be successfully used for preparing an affinity column, producing GATA-1-specific rabbit polyclonal antibodies, and studying DNA-protein and protein-protein interactions.     

An improved protocol for electroporation of Oenococcus oeni ATCC BAA‐1163 using ethanol as immediate membrane fluidizing agent

Aims: To finalize an effective and reproducible electroporation procedure to transform Oenococcus oeni ATCC BAA‐1163 strain. Methods and Results: The vector pGID052 was selected to optimize the electroporation procedure. Transformation efficiency was 5·8 × 10³ per μg of DNA. Transformation was improved when competent cells were prepared with exponential phase cultures; optimum electroporation parameters were an electric pulse of 12·5 kV cm^?1, under a resistance of 200 Ω and the presence of 10% (v/v) ethanol in the electroporation buffer (EPB). Conclusions: An effective protocol to transform O. oeni ATCC BAA‐1163 strain by electroporation has been obtained by addition of ethanol to the EPB. A heterologous expression was obtained in O. oeni ATCC BAA‐1163 by introducing a recombinant vector encoding a truncated form of ClpL2 protein. Significance and Impact of the Study: This is the first report of a successful electroporation of O. oeni ATCC BAA‐1163. The major improvement was the addition of ethanol to the EPB, which has never been reported before as means of enhancing the incorporation of foreign DNA molecules into prokaryote cells by electroporation. This method constitutes a useful tool for the genetic study of this lactic bacterium.     

The cytotoxic norditerpene dilactones of Podocarpus milanjianus and Podocarpus sellowii

The cytotoxic norditerpene dilactones nagilactone F and its new congener nagilactone G have been isolated from the bark constituents of Podocarpus milanjianus and Podocarpus sellowii. The diterpenes totarol, 19-oxototarol and macrophyllic acid were also isolated.