Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).

Transient receptor potential ion channels V4 and A1 contribute to pancreatitis pain in mice.

The mechanisms of pancreatic pain, a cardinal symptom of pancreatitis, are unknown. Proinflammatory agents that activate transient receptor potential (TRP) channels in nociceptive neurons can cause neurogenic inflammation and pain. We report a major role for TRPV4, which detects osmotic pressure and arachidonic acid metabolites, and TRPA1, which responds to 4-hydroxynonenal and cyclopentenone prostaglandins, in pancreatic inflammation and pain in mice. Immunoreactive TRPV4 and TRPA1 were detected in pancreatic nerve fibers and in dorsal root ganglia neurons innervating the pancreas, which were identified by retrograde tracing. Agonists of TRPV4 and TRPA1 increased intracellular Ca(2+) concentration ([Ca(2+)](i)) in these neurons in culture, and neurons also responded to the TRPV1 agonist capsaicin and are thus nociceptors. Intraductal injection of TRPV4 and TRPA1 agonists increased c-Fos expression in spinal neurons, indicative of nociceptor activation, and intraductal TRPA1 agonists also caused pancreatic inflammation. The effects of TRPV4 and TRPA1 agonists on [Ca(2+)](i), pain and inflammation were markedly diminished or abolished in trpv4 and trpa1 knockout mice. The secretagogue cerulein induced pancreatitis, c-Fos expression in spinal neurons, and pain behavior in wild-type mice. Deletion of trpv4 or trpa1 suppressed c-Fos expression and pain behavior, and deletion of trpa1 attenuated pancreatitis. Thus TRPV4 and TRPA1 contribute to pancreatic pain, and TRPA1 also mediates pancreatic inflammation. Our results provide new information about the contributions of TRPV4 and TRPA1 to inflammatory pain and suggest that channel antagonists are an effective therapy for pancreatitis, when multiple proinflammatory agents are generated that can activate and sensitize these channels.

Protease-activated receptors: mechanisms by which proteases sensitize TRPV channels to induce neurogenic inflammation and pain

Proteolytic enzymes comprise approximately 2 percent of the human genome [1]. Given their abundance, it is not surprising that proteases have diverse biological functions, ranging from the degradation of proteins in lysosomes to the control of physiological processes such as the coagulation cascade. However, a subset of serine proteases (possessing serine residues within their catalytic sites), which may be soluble in the extracellular fluid or tethered to the plasma membrane, are signaling molecules that can specifically regulate cells by cleaving protease-activated receptors (PARs), a family of four G-protein-coupled receptors (GPCRs). These serine proteases include members of the coagulation cascade (e.g., thrombin, factor VIIa, and factor Xa), proteases from inflammatory cells (e.g., mast cell tryptase, neutrophil cathepsin G), and proteases from epithelial tissues and neurons (e.g., trypsins). They are often generated or released during injury and inflammation, and they cleave PARs on multiple cell types, including platelets, endothelial and epithelial cells, myocytes, fibroblasts, and cells of the nervous system. Activated PARs regulate many essential physiological processes, such as hemostasis, inflammation, pain, and healing. These proteases and their receptors have been implicated in human disease and are potentially important targets for therapy. Proteases and PARs participate in regulating most organ systems and are the subject of several comprehensive reviews [2, 3]. Within the central and peripheral nervous systems, proteases and PARs can control neuronal and astrocyte survival, proliferation and morphology, release of neurotransmitters, and the function and activity of ion channels, topics that have also been comprehensively reviewed [4, 5]. This chapter specifically concerns the ability of PARs to regulate TRPV channels of sensory neurons and thereby affect neurogenic inflammation and pain transmission [6, 7].

Flood risk and climate change: global and regional perspectives

A holistic perspective on changing rainfall-driven flood risk is provided for the late 20th and early 21st centuries. Economic losses from floods have greatly increased, principally driven by the expanding exposure of assets at risk. It has not been possible to attribute rain-generated peak streamflow trends to anthropogenic climate change over the past several decades. Projected increases in the frequency and intensity of heavy rainfall, based on climate models, should contribute to increases in precipitation-generated local flooding (e.g. flash flooding and urban flooding). This article assesses the literature included in the IPCC SREX report and new literature published since, and includes an assessment of changes in flood risk in seven of the regions considered in the recent IPCC SREX report—Africa, Asia, Central and South America, Europe, North America, Oceania and Polar regions. Also considering newer publications, this article is consistent with the recent IPCC SREX assessment finding that the impacts of climate change on flood characteristics are highly sensitive to the detailed nature of those changes and that presently we have only low confidence1 in numerical projections of changes in flood magnitude or frequency resulting from climate change.

Evaluation of seven European aerosol optical depth retrieval algorithms for climate analysis

Satellite data are increasingly used to provide observation-based estimates of the effects of aerosols on climate. The Aerosol-cci project, part of the European Space Agency's Climate Change Initiative (CCI), was designed to provide essential climate variables for aerosols from satellite data. Eight algorithms, developed for the retrieval of aerosol properties using data from AATSR (4), MERIS (3) and POLDER, were evaluated to determine their suitability for climate studies. The primary result from each of these algorithms is the aerosol optical depth (AOD) at several wavelengths, together with the Ångström exponent (AE) which describes the spectral variation of the AOD for a given wavelength pair. Other aerosol parameters which are possibly retrieved from satellite observations are not considered in this paper. The AOD and AE (AE only for Level 2) were evaluated against independent collocated observations from the ground-based AERONET sun photometer network and against “reference” satellite data provided by MODIS and MISR. Tools used for the evaluation were developed for daily products as produced by the retrieval with a spatial resolution of 10 × 10 km2 (Level 2) and daily or monthly aggregates (Level 3). These tools include statistics for L2 and L3 products compared with AERONET, as well as scoring based on spatial and temporal correlations. In this paper we describe their use in a round robin (RR) evaluation of four months of data, one month for each season in 2008. The amount of data was restricted to only four months because of the large effort made to improve the algorithms, and to evaluate the improvement and current status, before larger data sets will be processed. Evaluation criteria are discussed. Results presented show the current status of the European aerosol algorithms in comparison to both AERONET and MODIS and MISR data. The comparison leads to a preliminary conclusion that the scores are similar, including those for the references, but the coverage of AATSR needs to be enhanced and further improvements are possible for most algorithms. None of the algorithms, including the references, outperforms all others everywhere. AATSR data can be used for the retrieval of AOD and AE over land and ocean. PARASOL and one of the MERIS algorithms have been evaluated over ocean only and both algorithms provide good results.

Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance

Evolution of resistance to drugs and pesticides poses a serious threat to human health and agricultural production. CYP51 encodes the target site of azole fungicides, widely used clinically and in agriculture. Azole resistance can evolve due to point mutations or overexpression of CYP51, and previous studies have shown that fungicide-resistant alleles have arisen by de novo mutation. Paralogs CYP51A and CYP51B are found in filamentous ascomycetes, but CYP51A has been lost from multiple lineages. Here, we show that in the barley pathogen Rhynchosporium commune, re-emergence of CYP51A constitutes a novel mechanism for the evolution of resistance to azoles. Pyrosequencing analysis of historical barley leaf samples from a unique long-term experiment from 1892 to 2008 indicates that the majority of the R. commune population lacked CYP51A until 1985, after which the frequency of CYP51A rapidly increased. Functional analysis demonstrates that CYP51A retains the same substrate as CYP51B, but with different transcriptional regulation. Phylogenetic analyses show that the origin of CYP51A far predates azole use, and newly sequenced Rhynchosporium genomes show CYP51A persisting in the R. commune lineage rather than being regained by horizontal gene transfer; therefore, CYP51A re-emergence provides an example of adaptation to novel compounds by selection from standing genetic variation.

Analysis of the bacterial communities present in lungs of patients with cystic fibrosis from American and British centers

The aim of this study was to determine whether geographical differences impact the composition of bacterial communities present in the airways of cystic fibrosis (CF) patients attending CF centers in the United States or United Kingdom. Thirty-eight patients were matched on the basis of clinical parameters into 19 pairs comprised of one U.S. and one United Kingdom patient. Analysis was performed to determine what, if any, bacterial correlates could be identified. Two culture-independent strategies were used: terminal restriction fragment length polymorphism (T-RFLP) profiling and 16S rRNA clone sequencing. Overall, 73 different terminal restriction fragment lengths were detected, ranging from 2 to 10 for U.S. and 2 to 15 for United Kingdom patients. The statistical analysis of T-RFLP data indicated that patient pairing was successful and revealed substantial transatlantic similarities in the bacterial communities. A small number of bands was present in the vast majority of patients in both locations, indicating that these are species common to the CF lung. Clone sequence analysis also revealed that a number of species not traditionally associated with the CF lung were present in both sample groups. The species number per sample was similar, but differences in species presence were observed between sample groups. Cluster analysis revealed geographical differences in bacterial presence and relative species abundance. Overall, the U.S. samples showed tighter clustering with each other compared to that of United Kingdom samples, which may reflect the lower diversity detected in the U.S. sample group. The impact of cross-infection and biogeography is considered, and the implications for treating CF lung infections also are discussed.

Structure and spectroelectrochemical response of arene− ruthenium and arene−osmium complexes with potentially hemilabile noninnocent ligands

Nine of the compounds [M(L2−)(p-cymene)] (M = Ru, Os, L2− = 4,6-di-tert-butyl-N-aryl-o-amidophenolate) were prepared and structurally characterized (Ru complexes) as coordinatively unsaturated, formally 16 valence electron species. On L2−-ligand based oxidation to EPR-active iminosemiquinone radical complexes, the compounds seek to bind a donor atom (if available) from the N-aryl substituent, as structurally certified for thioether and selenoether functions, or from the donor solvent. Simulated cyclic voltammograms and spectroelectrochemistry at ambient and low temperatures in combination with DFT results confirm a square scheme behavior (ECEC mechanism) involving the Ln ligand as the main electron transfer site and the metal with fractional (δ) oxidation as the center for redox-activated coordination. Attempts to crystallize [Ru(Cym)(QSMe)](PF6) produced single crystals of [RuIII(QSMe •−)2](PF6) after apparent dissociation of the arene ligand.

Climate and carbon cycle changes from 1850 to 2100 in MPI-ESM simulations for the Coupled Model Intercomparison Project phase 5

The new Max-Planck-Institute Earth System Model (MPI-ESM) is used in the Coupled Model Intercomparison Project phase 5 (CMIP5) in a series of climate change experiments for either idealized CO2-only forcing or forcings based on observations and the Representative Concentration Pathway (RCP) scenarios. The paper gives an overview of the model configurations, experiments related forcings, and initialization procedures and presents results for the simulated changes in climate and carbon cycle. It is found that the climate feedback depends on the global warming and possibly the forcing history. The global warming from climatological 1850 conditions to 2080–2100 ranges from 1.5°C under the RCP2.6 scenario to 4.4°C under the RCP8.5 scenario. Over this range, the patterns of temperature and precipitation change are nearly independent of the global warming. The model shows a tendency to reduce the ocean heat uptake efficiency toward a warmer climate, and hence acceleration in warming in the later years. The precipitation sensitivity can be as high as 2.5% K−1 if the CO2 concentration is constant, or as small as 1.6% K−1, if the CO2 concentration is increasing. The oceanic uptake of anthropogenic carbon increases over time in all scenarios, being smallest in the experiment forced by RCP2.6 and largest in that for RCP8.5. The land also serves as a net carbon sink in all scenarios, predominantly in boreal regions. The strong tropical carbon sources found in the RCP2.6 and RCP8.5 experiments are almost absent in the RCP4.5 experiment, which can be explained by reforestation in the RCP4.5 scenario.

A SNP-based consensus genetic map for synteny-based trait targeting in faba bean (Vicia faba L.)

Faba bean (Vicia faba L.) is a globally important nitrogen-fixing legume, which is widely grown in a diverse range of environments. In this work, we mine and validate a set of 845 SNPs from the aligned transcriptomes of two contrasting inbred lines. Each V. faba SNP is assigned by BLAST analysis to a single Medicago orthologue. This set of syntenically anchored polymorphisms were then validated as individual KASP assays, classified according to their informativeness and performance on a panel of 37 inbred lines, and the best performing 757 markers used to genotype six mapping populations. The six resulting linkage maps were merged into a single consensus map on which 687 SNPs were placed on six linkage groups, each presumed to correspond to one of the six V. faba chromosomes. This sequence-based consensus map was used to explore synteny with the most closely-related crop species, lentil, and the most closely related fully sequenced genome, Medicago. Large tracts of uninterrupted colinearity were found between faba bean and Medicago, making it relatively straightforward to predict gene content and order in mapped genetic interval. As a demonstration of this, we mapped a flower colour gene to a 2 cM interval of Vf chromosome 2 which was highly collinear with Mt3. The obvious candidate gene from 77 gene models in the collinear Medicago chromosome segment was the previously characterized MtWD40-1 gene (Mt3g092830, Mt3g092840) controlling anthocyanin production in Medicago and re-sequencing of the Vf orthologue showed a putative causative deletion of the entire 5’ end of the gene.

Morphological characterization of periodontium-derived human stem cells

The aim of this study has been to characterize adult human somatic periodontium-derived stem cells (PDSCS) isolated from human periodontium and to follow their differentiation after cell culture. PDSCS were isolated from human periodontal tissue and cultured as spheres in serum-free medium. After 10 days the primary spheres were dissociated and the secondary spheres sub-cultured for another 1-2 weeks. Cells from different time points were analyzed, and immunohistochemical and electron microscopic investigations carried out. Histological analysis showed differentiation of spheres deriving from the PDSCS with central production of extracellular matrix beginning 3 days after sub-culturing. Isolated PDSCS developed pseudopodia which contained actin. Tubulin was found in the central portion of the cells. Pseudopodia between different cells anastomosed, indicating intercellular transport. Immunostaining for osteopontin demonstrated a positive reaction in primary spheres and within extracellular matrix vesicles after sub-culturing. In cell culture under serum-free conditions human PDSCS form spheres which are capable of producing extracellular matrix. Further investigations have do be carried out to investigate the capability of these cells to differentiate into osteogenic progenitor cells.

An intronic G run within HIV-1 intron 2 is critical for splicing regulation of vif mRNA

Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3' splice site (3'ss) A1 but lack splicing at 5'ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3'ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3'ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5'ss D2. Here we show that an intronic G run (G(I2)-1) represses the use of a second 5'ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of G(I2)-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here.