MMP-9, TIMP-1 and inflammatory cells in sputum from COPD patients during exacerbation

Background

Irreversible airflow obstruction in Chronic Obstructive Pulmonary Disease (COPD) is thought to result from airway remodelling associated with aberrant inflammation. Patients who experience frequent episodes of acute deterioration in symptoms and lung function, termed exacerbations, experience a faster decline in their lung function, and thus over time greater disease severity However the mechanisms by which these episodes may contribute to decreased lung function are poorly understood.This study has prospectively examined changes in sputum levels of inflammatory cells, MMP-9 and TIMP-1 during exacerbations comparing with paired samples taken prior to exacerbation.

Methods

Nineteen COPD patients ((median, [IQR]) age 69 [63 to 74], forced expiratory volume in one second (FEV1) 1.0 [0.9 to1.2], FEV1% predicted 37.6 [27.3 to 46.2]) provided sputa at exacerbation. Of these, 12 were paired with a samples collected when the patient was stable, a median 4 months [2 to 8 months] beforehand.

Results

MMP-9 levels increased from 10.5 μg/g [1.2 to 21.1] prior to exacerbation to 17.1 μg/g [9.3 to 48.7] during exacerbation (P < 0.01). TIMP-1 levels decreased from 3.5 μg/g [0.6 to 7.8] to 1.5 μg/g [0.3 to 4.9] (P = 0.16). MMP-9/TIMP-1 Molar ratio significantly increased from 0.6 [0.2 to 1.1] to 3.6 [2.0 to 25.3] (P < 0.05). Neutrophil, eosinophil and lymphocyte counts all showed significant increase during exacerbation compared to before (P < 0.05). Macrophage numbers remained level. MMP-9 levels during exacerbation showed highly significant correlation with both neutrophil and lymphocyte counts (Rho = 0.7, P < 0.01).

Conclusion

During exacerbation, increased inflammatory burden coincides with an imbalance of the proteinase MMP-9 and its cognate inhibitor TIMP-1. This may suggest a pathway connecting frequent exacerbations with lung function decline.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Phylogenetic inference: how much evolutionary history is knowable?

In order to reconstruct phylogenetic trees from extremely dissimilar sequences it is necessary to estimate accurately the extent of sequence divergence. In this paper a new method of sequence analysis, Markov triple analysis, is developed for determining the relative frequencies of nucleotide substitutions within the three branches of a three-taxon dendrogram. Assuming that nucleotide sites are independently and identically distributed and assuming a Markov model for nucleotide (or protein) evolution, it is shown that the unique Markov matrices can be reconstructed given only the joint probability distribution relating three taxa. (In the much simpler case involving only two taxa and two character states, Markov matrices can also be reconstructed, provided symmetry assumptions are placed on the elements of the matrices.) The method is illustrated using sequence data from the combined first and second codon positions derived from complete human, mouse, and cow mitochondrial sequences.      

DNA hybridization evidence for the principal lineages of hummingbirds (Aves:Trochilidae)

The spectacular evolutionary radiation of hummingbirds (Trochilidae) has served as a model system for many biological studies. To begin to provide a historical context for these investigations, we generated a complete matrix of DNA hybridization distances among 26 hummingbirds and an outgroup swift (Chaetura pelagica) to determine the principal hummingbird lineages. FITCH topologies estimated from symmetrized delta TmH-C values and subjected to various validation methods (bootstrapping, weighted jackknifing, branch length significance) indicated a fundamental split between hermit (Eutoxeres aquila, Threnetes ruckeri; Phaethornithinae) and nonhermit (Trochilinae) hummingbirds, and provided strong support for six principal nonhermit clades with the following branching order: (1) a predominantly lowland group comprising caribs (Eulampis holosericeus) and relatives (Androdon aequatorialis and Heliothryx barroti) with violet-ears (Colibri coruscans) and relatives (Doryfera ludovicae); (2) an Andean-associated clade of highly polytypic taxa (Eriocnemis, Heliodoxa, and Coeligena); (3) a second endemic Andean clade (Oreotrochilus chimborazo, Aglaiocercus coelestis, and Lesbia victoriae) paired with thorntails (Popelairia conversii); (4) emeralds and relatives (Chlorostilbon mellisugus, Amazilia tzacatl, Thalurania colombica, Orthorhyncus cristatus and Campylopterus villaviscensio); (5) mountain-gems (Lampornis clemenciae and Eugenes fulgens); and (6) tiny bee-like forms (Archilochus colubris, Myrtis fanny, Acestrura mulsant, and Philodice mitchellii). Corresponding analyses on a matrix of unsymmetrized delta values gave similar support for these relationships except that the branching order of the two Andean clades (2, 3 above) was unresolved. In general, subsidiary relationships were consistent and well supported by both matrices, sometimes revealing surprising associations between forms that differ dramatically in plumage and bill morphology. Our results also reveal some basic aspects of hummingbird ecologic and morphologic evolution. For example, most of the diverse endemic Andean assemblage apparently comprises two genetically divergent clades, whereas the majority of North American hummingbirds belong a single third clade. Genetic distances separating some morphologically distinct genera (Oreotrochilus, Aglaiocercus, Lesbia; Myrtis, Acestrura, Philodice) were no greater than among congeneric (Coeligena) species, indicating that, in hummingbirds, morphological divergence does not necessarily reflect level of genetic divergence.      

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

Differences in the carbon tetrachloride-induced damage to components of the smooth and rough endoplasmic reticulum from rat liver

There is a higher activity of ethyl morphine N-demethylase (EM-ase) and cytochrome P-450 (P-450) reductase as well as higher P-450 content in the smooth endoplasmic reticulum (SER) than in the rough endoplasmic reticulum (RER). The extent of the irreversible binding of the¹⁴C from¹⁴CCl₄ to lipids and proteins, as well as the CCl₄-induced destruction of P-450 is more intense in SER than in RER while the opposite was found for glucose 6-phosphatase (G6P-ase) destruction. CCl₄-induced lipid peroxidation is as intense in SER as is in RER.¹⁴C from¹⁴CCl₄ gets irreversibly bound to ribosomal proteins.     

Focus on Weed Control: Dual Function of the Cytochrome P450 CYP76 Family from Arabidopsis thaliana in the Metabolism of Monoterpenols and Phenylurea Herbicides

Comparative genomics analysis unravels lineage-specific bursts of gene duplications related to the emergence of specialized pathways. The CYP76C subfamily of cytochrome P450 enzymes is specific to Brassicaceae. Two of its members were recently associated with monoterpenol metabolism. This prompted us to investigate the CYP76C subfamily genetic and functional diversification. Our study revealed high rates of CYP76C gene duplication and loss in Brassicaceae, suggesting the association of the CYP76C subfamily with species-specific adaptive functions. Gene differential expression and enzyme functional specialization in Arabidopsis thaliana, including metabolism of different monoterpenols and formation of different products, support this hypothesis. In addition to linalool metabolism, CYP76C1, CYP76C2, and CYP76C4 metabolized herbicides belonging to the class of phenylurea. Their ectopic expression in the whole plant conferred herbicide tolerance. CYP76Cs from A. thaliana. thus provide a first example of promiscuous cytochrome P450 enzymes endowing effective metabolism of both natural and xenobiotic compounds. Our data also suggest that the CYP76C gene family provides a suitable genetic background for a quick evolution of herbicide resistance.Although extensive monoterpenol (especially linalool) oxidative metabolism has been described in many plant species, leading to fragrant and bioactive compounds as diverse as alcohols, aldehydes, acids, and epoxides (Williams et al., 1982; Matich et al., 2003, 2011; Luan et al., 2005, 2006; Ginglinger et al., 2013), pyranoid or furanoid linalool derivatives (Pichersky et al., 1994; Raguso and Pichersky, 1999), and geraniol-derived iridoids and secoiridoids (Dinda et al., 2007a, 2007b, 2011; Tundis et al., 2008), limited information is available on the enzymes generating these oxygenated compounds. Involvement of a cytochrome P450 (P450) enzyme extracted from Vinca rosea (now renamed Catharanthus roseus) in the hydroxylation of geraniol and nerol was suggested as early as 1976 (Madyastha et al., 1976). The first plant P450 gene to be isolated, CYP71A1 from avocado (Persea americana) fruit, was later shown to encode an enzyme with geraniol/nerol epoxidase activity (Hallahan et al., 1992, 1994). To our knowledge, a connection with compounds formed in the fruit has not yet been established. The geraniol 8-hydroxylase (often named geraniol 10-hydroxylase) CYP76B6, involved in the biosynthesis of secoiridoids and monoterpene indole alkaloid anticancer drugs in C. roseus, was found to belong to the CYP76 family in 2001 (Collu et al., 2001). The catalytic function of this enzyme was recently revised, and was shown to include a second oxidation activity, the conversion of 8-hydroxygeraniol into 8-oxogeraniol (Höfer et al., 2013). The same work also revealed a geraniol 8- and 9-hydroxylase activity of CYP76C4 from Arabidopsis thaliana. More recently, another CYP76 enzyme (CYP76A226) from C. roseus was found to metabolize oxidized geraniol derivatives and to have an iridoid oxidase activity, catalyzing the triple oxygenation of cis-trans-nepetalactol into 7-deoxyloganetic acid for the biosynthesis of secoiridoids and terpene indole alkaloids (Miettinen et al., 2014; Salim et al., 2014). Not all CYP76 enzymes seem to be devoted to the metabolism of monoterpenols. In most cases, however, CYP76s seem to be involved in terpenoid metabolism. CYP76Ms from monocots were found to metabolize diterpenoids for the synthesis of antifungal phytocassanes (Swaminathan et al., 2009; Wang et al., 2012; Wu et al., 2013), CYP76AH1 from Salvia miltiorhizza and its ortholog CYP76AH4 from rosemary (Rosmarinus officinalis) were shown to hydroxylate the norditerpene abietatriene in the pathway to labdane-related compounds (Zi and Peters, 2013), whereas CYP76Fs from sandalwood (Santalum album) were found to hydroxylate the sesquiterpenes santalene and bergamotene (Diaz-Chavez et al., 2013). CYP76B1 from Helianthus tuberosus was, however, found to metabolize herbicides belonging to the class of phenylurea (Robineau et al., 1998; Didierjean et al., 2002), but its physiological function was not reported. Other P450s from soybean (Glycine max; CYP71A10; Siminszky et al., 1999) or tobacco (Nicotiana tabacum; CYP71A11 and CYP81B1; Yamada et al., 2000) were also reported to metabolize phenylurea, but their physiological function was not investigated.A. thaliana ecotype Columbia-0 (Col-0) emits no geraniol and only tiny amounts of linalool, and extensive volatile profiling of different tissues detected only minor amounts of lilac aldehydes (oxygenated linalool derivatives; Rohloff and Bones, 2005). However, ectopic expression of a linalool/nerolidol synthase of strawberry (Fragaria × anannasa cv Elsanta) revealed a potentially efficient oxidative linalool metabolism in A. thaliana rosette leaves (Aharoni et al., 2003). Only recent work started to explore linalool metabolism in A. thaliana, which was found mainly localized in the flowers (Ginglinger et al., 2013). This work demonstrated the existence of two linalool synthases producing different enantiomers, and the concomitant involvement of two P450 enzymes, CYP76C3 and CYP71B31, with predominance of CYP76C3, in linalool oxidation. It also suggested the presence of partially redundant enzymes that may contribute to floral linalool metabolism.A family of eight CYP76 genes is detected in the A. thaliana genome. We report here an evolutionary and functional analysis of this family. We show that members of the CYP76C subfamily, when successfully expressed in yeast (Saccharomyces cerevisiae), all metabolize monoterpenols with different substrate specificities. Although CYP76Cs seem specific to Brassicaceae, they share common functions with CYP76s from other plants, such as CYP76B1 from H. tuberosus and CYP76B6 from C. roseus. These functions include not only monoterpenol oxidation, but also metabolism and detoxification of herbicides belonging to the class of phenylurea. Because of this property, CYP76Cs can be used simultaneously for monoterpenol oxidation and as selectable markers for plant transformation.     

Genomic characterisation of the effector complement of the potato cyst nematode Globodera pallida

Background

The potato cyst nematode Globodera pallida has biotrophic interactions with its host. The nematode induces a feeding structure – the syncytium – which it keeps alive for the duration of the life cycle and on which it depends for all nutrients required to develop to the adult stage. Interactions of G. pallida with the host are mediated by effectors, which are produced in two sets of gland cells. These effectors suppress host defences, facilitate migration and induce the formation of the syncytium.

Results

The recent completion of the G. pallida genome sequence has allowed us to identify the effector complement from this species. We identify 128 orthologues of effectors from other nematodes as well as 117 novel effector candidates. We have used in situ hybridisation to confirm gland cell expression of a subset of these effectors, demonstrating the validity of our effector identification approach. We have examined the expression profiles of all effector candidates using RNAseq; this analysis shows that the majority of effectors fall into one of three clusters of sequences showing conserved expression characteristics (invasive stage nematode only, parasitic stage only or invasive stage and adult male only). We demonstrate that further diversity in the effector pool is generated by alternative splicing. In addition, we show that effectors target a diverse range of structures in plant cells, including the peroxisome. This is the first identification of effectors from any plant pathogen that target this structure.

Conclusion

This is the first genome scale search for effectors, combined to a life-cycle expression analysis, for any plant-parasitic nematode. We show that, like other phylogenetically unrelated plant pathogens, plant parasitic nematodes deploy hundreds of effectors in order to parasitise plants, with different effectors required for different phases of the infection process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-923) contains supplementary material, which is available to authorized users.