Spatial and temporal variations in the Annual Pollen Index recorded by sites belonging to the Portuguese Aerobiology Network

This study presents the findings of a 10-year survey carried out by the Portuguese Aerobiology Network (RPA) at seven pollen-monitoring stations: five mainland stations (Oporto, Coimbra, Lisbon, Évora and Portimão) and two insular stations [Funchal (Madeira archipelago) and Ponta Delgada (Azores archipelago)]. The main aim of the study was to examine spatial and temporal variations in the Annual Pollen Index (API) with particular focus on the most frequently recorded pollen types. Pollen monitoring (2003–2012) was carried out using Hirst-type volumetric spore traps, following the minimum recommendations proposed by the European Aerobiology Society Working Group on Quality Control. Daily pollen data were examined for similarities using the Kruskal–Wallis nonparametric test and multivariate regression trees. Simple linear regression analysis was used to describe trends in API. The airborne pollen spectrum at RPA stations is dominated by important allergenic pollen types such as Poaceae, Olea and Urticaceae. Statistically significant differences were witnessed in the API recorded at the seven stations. Mean API is higher in the southern mainland cities, e.g. Évora, Lisbon and Portimão, and lower in insular and littoral cities. There were also a number of significant trends in API during the 10-year study. This report identifies spatial and temporal variations in the amount of airborne pollen recorded annually in the Portuguese territory. There were also a number of significant changes in API, but no general increases in the amount of airborne pollen.     

Epitope characterization of anti-JAM-A antibodies using orthogonal mass spectrometry and surface plasmon resonance approaches

Junctional adhesion molecule-A (JAM-A) is an adherens and tight junction protein expressed by endothelial and epithelial cells and associated with cancer progression. We present here the extensive characterization of immune complexes involving JAM-A antigen and three monoclonal antibodies (mAbs), including hz6F4-2, a humanized version of anti-tumoral 6F4 mAb identified by a functional and proteomic approach in our laboratory. A specific workflow that combines orthogonal approaches has been designed to determine binding stoichiometries along with JAM-A epitope mapping determination at high resolution for these three mAbs. Native mass spectrometry experiments revealed different binding stoichiometries and affinities, with two molecules of JAM-A being able to bind to hz6F4-2 and F11 Fab, while only one JAM-A was bound to J10.4. Surface plasmon resonance indirect competitive binding assays suggested epitopes located in close proximity for hz6F4-2 and F11. Finally, hydrogen-deuterium exchange mass spectrometry was used to precisely identify epitopes for all mAbs. The results obtained by orthogonal biophysical approaches showed a clear correlation between the determined epitopes and JAM-A binding characteristics, allowing the basis for molecular recognition of JAM-A by hz6F4-2 to be definitively established for the first time. Taken together, our results highlight the power of MS-based structural approaches for epitope mapping and mAb conformational characterization.     

Vanadium-dependent iodoperoxidases in Laminaria digitata, a novel biochemical function diverging from brown algal bromoperoxidases

The brown alga Laminaria digitata features a distinct vanadium-dependent iodoperoxidase (vIPO) activity, which has been purified to electrophoretic homogeneity. Steady-state analyses at pH 6.2 are reported for vIPO (K _m ^I– =2.5 mM; k _cat ^I– =462 s^–1) and for the previously characterised vanadium-dependent bromoperoxidase in L. digitata (K _m ^I– =18.1 mM; k _cat ^I– =38 s^–1). Although the vIPO enzyme specifically oxidises iodide, competition experiments with halides indicate that bromide is a competitive inhibitor with respect to the fixation of iodide. A full-length complementary ANA (cDNA) was cloned and shown to be actively transcribed in L. digitata and to encode the vIPO enzyme. Mass spectrometry analyses of tryptic digests of vIPO indicated the presence of at least two very similar proteins, in agreement with Southern analyses showing that vIPOs are encoded by a multigenic family in L. digitata. Phylogenetic analyses indicated that vIPO shares a close common ancestor with brown algal vanadium-dependent bromoperoxidases. Based on a three-dimensional structure model of the vIPO active site and on comparisons with those of other vanadium-dependent haloperoxidases, we propose a hypothesis to explain the evolution of strict specificity for iodide in L. digitata vIPO.The nucleotide sequence reported in this paper has been submitted to the EBI Data Bank with accession no. AJ619804.     

The human spindle assembly checkpoint protein Bub3 is required for the establishment of efficient kinetochore-microtubule attachments

The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.     

The evolutionary history of sharp- and blunt-snouted lenok (<Emphasis Type="Italic">Brachymystax lenok</Emphasis> (Pallas, 1773)) and its implications for the paleo-hydrological history of Siberia

Background  

Broad-scale phylogeographic studies of freshwater organisms provide not only an invaluable framework for understanding the evolutionary history of species, but also a genetic imprint of the paleo-hydrological dynamics stemming from climatic change. Few such studies have been carried out in Siberia, a vast region over which the extent of Pleistocene glaciation is still disputed. Brachymystax lenok is a salmonid fish distributed throughout Siberia, exhibiting two forms hypothesized to have undergone extensive range expansion, genetic exchange, and multiple speciation. A comprehensive phylogeographic investigation should clarify these hypotheses as well as provide insights on Siberia's paleo-hydrological stability.     

Anchored plasticity opens doors for selective inhibitor design in nitric oxide synthase

Nitric oxide synthase (NOS) enzymes synthesize nitric oxide, a signal for vasodilatation and neurotransmission at low concentrations and a defensive cytotoxin at higher concentrations. The high active site conservation among all three NOS isozymes hinders the design of selective NOS inhibitors to treat inflammation, arthritis, stroke, septic shock and cancer. Our crystal structures and mutagenesis results identified an isozyme-specific induced-fit binding mode linking a cascade of conformational changes to a new specificity pocket. Plasticity of an isozyme-specific triad of distant second- and third-shell residues modulates conformational changes of invariant first-shell residues to determine inhibitor selectivity. To design potent and selective NOS inhibitors, we developed the anchored plasticity approach: anchor an inhibitor core in a conserved binding pocket, then extend rigid bulky substituents toward remote specificity pockets, which become accessible upon conformational changes of flexible residues. This approach exemplifies general principles for the design of selective enzyme inhibitors that overcome strong active site conservation.     

Synthesis and structure based optimization of novel Akt inhibitors

Based on a high throughput screening hit, pyrrolopyrimidine inhibitors of the Akt kinase are explored. X-ray co-crystal structures of two lead series results in the understanding of key binding interactions, the design of new lead series, and enhanced potency. The syntheses of these series and their biological activities are described. Spiroindoline 13j is found to have an Akt1 kinase IC(50) of 2.4+/-0.6 nM, Akt cell potency of 50+/-19 nM, and provides 68% inhibition of tumor growth in a mouse xenograft model (50 mg/kg, qd, po).     

Road building, land use and climate change: prospects for environmental governance in the Amazon

Some coupled land-climate models predict a dieback of Amazon forest during the twenty-first century due to climate change, but human land use in the region has already reduced the forest cover. The causation behind land use is complex, and includes economic, institutional, political and demographic factors. Pre-eminent among these factors is road building, which facilitates human access to natural resources that beget forest fragmentation. While official government road projects have received considerable attention, unofficial road building by interest groups is expanding more rapidly, especially where official roads are being paved, yielding highly fragmented forest mosaics. Effective governance of natural resources in the Amazon requires a combination of state oversight and community participation in a 'hybrid' model of governance. The MAP Initiative in the southwestern Amazon provides an example of an innovative hybrid approach to environmental governance. It embodies a polycentric structure that includes government agencies, NGOs, universities and communities in a planning process that links scientific data to public deliberations in order to mitigate the effects of new infrastructure and climate change.     

Pulsed-field gel electrophoresis (PFGE) analysis of Listeria monocytogenes isolates from different sources and geographical origins and representative of the twelve serovars

Multiplex-PCR (MPCR) serogrouping and pulsed-field gel electrophoresis (PFGE) subtyping analysis are currently used by several public and private laboratories for the characterization of Listeria monocytogenes. In this study a set of 80 L. monocytogenes isolates belonging to the twelve serovars was used to investigate (i) the typeability of the rare serovars, (ii) the ability of PFGE analysis with ApaI and AscI to differentiate serovars within MPCR serogroups and (iii) the association of molecular types with the specific source or geographical origin of the isolates. With the exception of three isolates (rare serovars 4a and 4c) that were not amenable to restriction with ApaI, all the other analyzed isolates were subtyped by both enzymes. PFGE discriminated the 80 isolates into 62 combined ApaI and AscI PFGE patterns (pulsotypes), but could not differentiate serovars within MPCR serogroups, in which isolates from different serovars displaying the same pulsotype were found. Clustering analysis suggests that for some pulsotypes grouping according to Portuguese origin or source can be suggested. On the other hand, some L. monocytogenes clones are widely distributed. Two pulsotypes from Portuguese human isolates were identical to the ones displayed by human outbreak clones in the UK and in the USA and Switzerland, respectively, although they were not temporally matched. Computer-assisted data analysis of large and diverse PFGE type databases will improve the correct interpretation of subtyping data in epidemiological studies and in tracing routes and sources of contamination in the food industry.