The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease

Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications

Putative resistance gene markers associated with quantitative trait loci for fire blight resistance in Malus 'Robusta 5' accessions

Background Breeding of fire blight resistant scions and rootstocks is a goal of several international apple breeding programs, as options are limited for management of this destructive disease caused by the bacterial pathogen Erwinia amylovora. A broad, large-effect quantitative trait locus (QTL) for fire blight resistance has been reported on linkage group 3 of Malus ‘Robusta 5’. In this study we identified candidate gene markers for fire blight resistance by integrating further genetic mapping studies with bioinformatics analysis of transcript profiling data and genome sequence databases. Results When several defined E.amylovora strains were used to inoculate three progenies from 62 international breeding programs, all with ‘Robusta 5’ as a common parent, two distinct QTLs were detected on linkage group 3, where only one had previously been mapped. In the New Zealand population, the proximal QTL co-located with SNP markers for a leucine-rich repeat, receptor-like protein (MxdRLP1) candidate resistance gene and a closely linked class 3 peroxidase gene. While the QTL detected in the German population was approximately 6 cM distal to this, directly below a SNP marker for a heat shock 90 family protein (HSP90). In the US population, the position of the LOD score peak on linkage group 3 was dependent upon the pathogen strains used for inoculation. One of the five MxdRLP1 alleles identified in fire blight resistant and susceptible cultivars was genetically associated with resistance and used to develop a high resolution melting PCR marker. A resistance QTL detected on linkage group 7 of the US population co-located with another HSP90 gene-family member and a WRKY transcription factor previously associated with fire blight resistance. However, this QTL was not observed in the New Zealand or German populations. Conclusions The results suggest that the upper region of ‘Robusta 5’ 76 linkage group 3 contains multiple genes contributing to fire blight resistance and that their contributions to resistance can vary depending upon pathogen virulence and other factors. Candidate gene mapping has proved a useful aid in defining these QTLs and developing markers for marker-assisted breeding of fire blight resistance

Phototriggered release of tetrapeptide AAPV from coumarinyl and pyrenyl cages

Ala-Ala-Pro-Val (AAPV) is a bioactive tetrapeptide that inhibits human neutrophil elastase (HNE), an enzyme involved in skin chronic inflammatory diseases like psoriasis. Caged derivatives of this peptide were prepared by proper N- and C-terminal derivatisation through a carbamate or ester linkage, respectively, with two photoactive moieties, namely 7-methoxycoumarin-2-ylmethyl and pyren-2-ylmethyl groups. These groups were chosen to assess the influence of the photosensitive group and the type of linkage in the controlled photorelease of the active molecule. The caged peptides were irradiated at selected wavelengths of irradiation (254, 300, and 350 nm), and the photolytic process was monitored by HPLC-UV. The results established the applicability of the tested photoactive groups for the release of AAPV, especially for the derivative bearing the carbamate-linked pyrenylmethyl group, which displayed the shortest irradiation times for the release at the various wavelengths of irradiation (ca. 4 min at 254 nm, 8 min at 300 nm and 46 min at 350 nm).Thanks are due to the Fundação para a Ciência e Tecnologia (FCT, Portugal) for financial support to the portuguese NMR network (PTNMR, Bruker Avance III 400- Univ. Minho), FCT and FEDER (European Fund for Regional Development)- COMPETE-QREN-EU for financial support through the Chemistry Research Centre of the University of Minho (Ref. UID/QUI/00686/2013 and UID/QUI/0686/2016). A PhD grant to A.M.S. (SFRH/BD/80813/2011) is also acknowledged.info:eu-repo/semantics/publishedVersio

Identification of Pyrus Single Nucleotide Polymorphisms (SNPs) and Evaluation for Genetic Mapping in European Pear and Interspecific Pyrus Hybrids

We have used new generation sequencing (NGS) technologies to identify single nucleotide polymorphism (SNP) markers from three European pear (Pyrus communis L.) cultivars and subsequently developed a subset of 1096 pear SNPs into high throughput markers by combining them with the set of 7692 apple SNPs on the IRSC apple Infinium® II 8K array. We then evaluated this apple and pear Infinium® II 9K SNP array for large-scale genotyping in pear across several species, using both pear and apple SNPs. The segregating populations employed for array validation included a segregating population of European pear (‘Old Home’בLouise Bon Jersey’) and four interspecific breeding families derived from Asian (P. pyrifolia Nakai and P. bretschneideri Rehd.) and European pear pedigrees. In total, we mapped 857 polymorphic pear markers to construct the first SNP-based genetic maps for pear, comprising 78% of the total pear SNPs included in the array. In addition, 1031 SNP markers derived from apple (13% of the total apple SNPs included in the array) were polymorphic and were mapped in one or more of the pear populations. These results are the first to demonstrate SNP transferability across the genera Malus and Pyrus. Our construction of high density SNP-based and gene-based genetic maps in pear represents an important step towards the identification of chromosomal regions associated with a range of horticultural characters, such as pest and disease resistance, orchard yield and fruit quality

Fire Blight resistance in apple/pear hybrids may be related to their genomic structure

Although Malus and Pyrus are closely related, with highly co-linear genomes, the two genera are characterised by many specific differences, including disease resistances, secondary metabolites, fruit texture, flavour and shape. Hence, intergeneric hybrids between apple and pear provide a unique germplasm resource for genetic analysis, as well as new cultivar development, using advanced genomic breeding strategies. Fire blight, caused by the Gram-negative bacterium Erwinia amylovora (Enterobacterales; Erwiniaceae), affects apple and pear production worldwide. A number of resistance loci against this disease have been located on genetic maps of both apple and pear.We investigated fire blight resistance in apple-pear hybrids, by studying 31 putative hybrids raised from a ‘Cox’s Orange Pippin’ x ‘Old Home’ cross at The New Zealand Institute for Plant and Food Research Limited. We inoculated up to eight replicates of each hybrid grafted on ‘M9’ rootstock and compared these with ‘Cox’s Orange Pippin’ and ‘Imperial Gala’ grafted on ‘M9’ rootstock and ‘Old Home’ and ‘Williams’ grafted with Pyrus calleryana, using the cut-leaf method (Maas Geesteranus and Heyting, 1981) for inoculation with E. amylovora (Ea 236 at 1 x 106 cfu/ml), as it is widely applied in both apple and pear. Disease progress was observed in the period from 2 to 6 weeks after inoculation. Level of disease was quantified by expressed necrosis length as a percentage of the total shoot length, both measured downwards from the point of inoculation. The result clearly showed that all of the 31 putative hybrids were resistant to fire blight, while the parents and controls exhibited the expected range of resistance and susceptibility according to previous work. Preliminary results using high-resolution melting marker analysis of the seedling genomes indicate there is a hybrid apple/pear genomic region on LG2, while LG7 is represented by apple DNA. Interestingly, fire blight resistance has been reported on LG2 of pear ‘Old Home’ (Montanari et al., 2016), while Khan et al. (2007) have located fire blight resistance on LG7 of the apple ‘Fiesta’, which is related by descent from ‘Cox’s Orange Pippin’.Our next step is to analyse recombination events during the crossing of apple and pear, using the IRSC 9K apple/pear SNP array: this will enable us to further investigate the relationship of these reported QTL resistances to fire blight infection within the genomic structure of our 31 apple/pear hybrid

A Functional Variant of Elafin With Improved Anti-inflammatory Activity for Pulmonary Inflammation

Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden