With more than 60% of the land farmed, with vulnerable freshwater and marine environments, and with one of the most intensive, export-oriented livestock sectors in the world, the nitrogen (N) ...pollution pressure from Danish agriculture is severe. Consequently, a series of policy action plans have been implemented since the mid 1980s with significant effects on the surplus, efficiency and environmental loadings of N. This paper reviews the policies and actions taken and their ability to mitigate effects of reactive N (Nr) while maintaining agricultural production. In summary, the average N-surplus has been reduced from approximately 170 kg N ha−1 yr−1 to below 100 kg N ha−1 yr−1 during the past 30 yrs, while the overall N-efficiency for the agricultural sector (crop + livestock farming) has increased from around 20-30% to 40-45%, the N-leaching from the field root zone has been halved, and N losses to the aquatic and atmospheric environment have been significantly reduced. This has been achieved through a combination of approaches and measures (ranging from command and control legislation, over market-based regulation and governmental expenditure to information and voluntary action), with specific measures addressing the whole N cascade, in order to improve the quality of ground- and surface waters, and to reduce the deposition to terrestrial natural ecosystems. However, there is still a major challenge in complying with the EU Water Framework and Habitats Directives, calling for new approaches, measures and technologies to mitigate agricultural N losses and control N flows.
Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide ...library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase-type plasminogen activator (uPA). We used X-ray crystal structure analysis, site-directed mutagenesis, liquid state NMR, surface plasmon resonance analysis, and isothermal titration calorimetry and wild type and engineered variants of murine and human uPA. We demonstrate that Arg6 inserts into the S1 specificity pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending on changes in both P1 - S1 and exosite interactions. Site-directed mutagenesis showed that exosite interactions, while still supporting high affinity binding, differed substantially between different uPA variants. Surprisingly, high affinity binding was facilitated by Ala-substitution of Asp9 of the peptide, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden.
•Sample of 71,999 roadway segments of rural two-lane roads was identified with GIS.•Models calculated separately for winter/non-winter and severe/slight accidents.•A non-monotonous link between risk ...and shoulder width categories.•An increased risk corresponds with increasing lane width categories.
This study estimates the effects of lane and shoulder widths on occurrence of head-on and single-vehicle accidents on rural two-lane undivided roads in Norway while considering the differences between winter and non-winter accidents and their severity levels. A matched case-control method was applied to calculate the odds ratios for lane and shoulder width categories, while controlling for the effects of AADT and adjusting for the effects of region, speed limit, segment length, share of long vehicles in AADT and horizontal alignment. The study used a sample of 71,999 roadway segments identified in GIS and 1886 related accidents recorded by the police in five-year period. The results suggest that it is relevant to consider winter and non-winter accidents as well as severe and slight accidents separately when studying the effects of lane and shoulder widths on the occurrence of head-on and single-vehicle accidents. When examining lane and shoulder widths for all related accidents, the lane widths 1.50–2.50 m and shoulder widths 0.50−0.75 m were relatively safer than other categories on Norwegian two-lane rural undivided roads.
Nucleic acid aptamer selection by systematic evolution of ligands by exponential enrichment (SELEX) has shown great promise for use in the development of research tools, therapeutics and diagnostics. ...Typically, aptamers are identified from libraries containing up to 10(16) different RNA or DNA sequences by 5-10 rounds of affinity selection towards a target of interest. Such library screenings can result in complex pools of many target-binding aptamers. New high-throughput sequencing techniques may potentially revolutionise aptamer selection by allowing quantitative assessment of the dynamic changes in the pool composition during the SELEX process and by facilitating large-scale post-SELEX characterisation. In the present study, we demonstrate how high-throughput sequencing of SELEX pools, before and after a single round of branched selection for binding to different target variants, can provide detailed information about aptamer binding sites, preferences for specific target conformations, and functional effects of the aptamers. The procedure was applied on a diverse pool of 2'-fluoropyrimidine-modified RNA enriched for aptamers specific for the serpin plasminogen activator inhibitor-1 (PAI-1) through five rounds of standard selection. The results demonstrate that it is possible to perform large-scale detailed characterisation of aptamer sequences directly in the complex pools obtained from library selection methods, thus without the need to produce individual aptamers.
Summary Background Despite successful treatment of the culprit artery lesion by primary percutaneous coronary intervention (PCI) with stent implantation, thrombotic embolisation occurs in some cases, ...which impairs the prognosis of patients with ST-segment elevation myocardial infarction (STEMI). We aimed to assess the clinical outcomes of deferred stent implantation versus standard PCI in patients with STEMI. Methods We did this open-label, randomised controlled trial at four primary PCI centres in Denmark. Eligible patients (aged >18 years) had acute onset symptoms lasting 12 h or less, and ST-segment elevation of 0·1 mV or more in at least two or more contiguous electrocardiographic leads or newly developed left bundle branch block. Patients were randomly assigned (1:1), via an electronic web-based system with permuted block sizes of two to six, to receive either standard primary PCI with immediate stent implantation or deferred stent implantation 48 h after the index procedure if a stabilised flow could be obtained in the infarct-related artery. The primary endpoint was a composite of all-cause mortality, hospital admission for heart failure, recurrent infarction, and any unplanned revascularisation of the target vessel within 2 years' follow-up. Patients, investigators, and treating clinicians were not masked to treatment allocation. We did analysis by intention to treat. This trial is registered with ClinicalTrials.gov , number NCT01435408. Findings Between March 1, 2011, and Feb 28, 2014, we randomly assigned 1215 patients to receive either standard PCI (n=612) or deferred stent implantation (n=603). Median follow-up time was 42 months (IQR 33–49). Events comprising the primary endpoint occurred in 109 (18%) patients who had standard PCI and in 105 (17%) patients who had deferred stent implantation (hazard ratio 0·99, 95% CI 0·76–1·29; p=0·92). Procedure-related myocardial infarction, bleeding requiring transfusion or surgery, contrast-induced nephopathy, or stroke occurred in 28 (5%) patients in the conventional PCI group versus 27 (4%) patients in the deferred stent implantation group, with no significant differences between groups. Interpretation In patients with STEMI, routine deferred stent implantation did not reduce the occurrence of death, heart failure, myocardial infarction, or repeat revascularisation compared with conventional PCI. Results from ongoing randomised trials might shed further light on the concept of deferred stenting in this patient population. Funding Danish Agency for Science, Technology and Innovation, and Danish Council for Strategic Research.
The membrane-bound serine protease matriptase belongs to a rare subset of serine proteases that display significant activity in the zymogen form. Matriptase is critically involved in epithelial ...differentiation and homeostasis, and insufficient regulation of its proteolytic activity directly causes onset and development of malignant cancer. There is strong evidence that the zymogen activity of matriptase is sufficient for its biological function(s). Activated matriptase is inhibited by the two Kunitz-type inhibitor domain-containing hepatocyte growth factor activator inhibitors 1 (HAI-1) and HAI-2, however, it remains unknown whether the activity of the matriptase zymogen is regulated. Using both purified proteins and a cell-based assay, we show that the catalytic activity of the matriptase zymogen towards a peptide-based substrate as well as the natural protein substrates, pro-HGF and pro-prostasin, can be inhibited by HAI-1 and HAI-2. Inhibition of zymogen matriptase by HAI-1 and HAI-2 appears similar to inhibition of activated matriptase and occurs at comparable inhibitor concentrations. This indicates that HAI-1 and HAI-2 interact with the active sites of zymogen and activated matriptase in a similar manner. Our results suggest that HAI-1 and HAI-2 regulate matriptase zymogen activity and thus may act as regulators of matriptase trans(auto)-activation. Due to the main localisation of HAI-2 in the ER and HAI-1 in the secretory pathway and on the cell surface, this regulation likely occurs both in the secretory pathway and on the plasma membrane. Regulation of an active zymogen form of a protease is a novel finding.
The spiny dogfish shark (Squalus acanthias) is one of the most commonly used cartilaginous fishes in biological research, especially in the fields of nitrogen metabolism, ion transporters and ...osmoregulation. Nonetheless, transcriptomic data for this organism is scarce. In the present study, a multi-tissue RNA-seq experiment and de novo transcriptome assembly was performed in four different spiny dogfish tissues (brain, liver, kidney and ovary), providing an annotated sequence resource. The characterization of the transcriptome greatly increases the scarce sequence information for shark species. Reads were assembled with the Trinity de novo assembler both within each tissue and across all tissues combined resulting in 362,690 transcripts in the combined assembly which represent 289,515 Trinity genes. BUSCO analysis determined a level of 87% completeness for the combined transcriptome. In total, 123,110 proteins were predicted of which 78,679 and 83,164 had significant hits against the SwissProt and Uniref90 protein databases, respectively. Additionally, 61,215 proteins aligned to known protein domains, 7,208 carried a signal peptide and 15,971 possessed at least one transmembrane region. Based on the annotation, 81,582 transcripts were assigned to gene ontology terms and 42,078 belong to known clusters of orthologous groups (eggNOG). To demonstrate the value of our molecular resource, we show that the improved transcriptome data enhances the current possibilities of osmoregulation research in spiny dogfish by utilizing the novel gene and protein annotations to investigate a set of genes involved in urea synthesis and urea, ammonia and water transport, all of them crucial in osmoregulation. We describe the presence of different gene copies and isoforms of key enzymes involved in this process, including arginases and transporters of urea and ammonia, for which sequence information is currently absent in the databases for this model species. The transcriptome assemblies and the derived annotations generated in this study will support the ongoing research for this particular animal model and provides a new molecular tool to assist biological research in cartilaginous fishes.
The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance, and inhibition of the complement ...alternative pathway is a subject of intense research. Here, we describe the nanobody hC3Nb1 that binds to multiple functional states of C3 with subnanomolar affinity. The nanobody causes a complete shutdown of alternative pathway activity in human and murine serum when present in concentrations comparable with that of C3, and hC3Nb1 is shown to prevent proconvertase assembly, as well as binding of the C3 substrate to C3 convertases. Our crystal structure of the C3b–hC3Nb1 complex and functional experiments demonstrate that proconvertase formation is blocked by steric hindrance between the nanobody and an Asn-linked glycan on complement factor B. In addition, hC3Nb1 is shown to prevent factor H binding to C3b, rationalizing its inhibition of factor I activity. Our results identify hC3Nb1 as a versatile, inexpensive, and powerful inhibitor of the alternative pathway in both human and murine in vitro model systems of complement activation.
The plasminogen (PLG) activation system is composed by a series of serine proteases, inhibitors and several binding proteins, which together control the temporal and spatial generation of the active ...serine protease plasmin. As this proteolytic system plays a central role in human physiology and pathophysiology it has been extensively studied in mammals. The serine proteases of this system are believed to originate from an ancestral gene by gene duplications followed by domain gains and deletions. However, the identification of ancestral forms in primitive chordates supporting these theories remains elusive. In addition, evolutionary studies of the non-proteolytic members of this system are scarce.
Our phylogenetic analyses place lamprey PLG at the root of the vertebrate PLG-group, while lamprey PLG-related growth factors represent the ancestral forms of the jawed-vertebrate orthologues. Furthermore, we find that the earliest putative orthologue of the PLG activator group is the hyaluronan binding protein 2 (HABP2) gene found in lampreys. The prime plasminogen activators (tissue- and urokinase-type plasminogen activator, tPA and uPA) first occur in cartilaginous fish and phylogenetic analyses confirm that all orthologues identified compose monophyletic groups to their mammalian counterparts. Cartilaginous fishes exhibit the most ancient vitronectin of all vertebrates, while plasminogen activator inhibitor 1 (PAI-1) appears for the first time in cartilaginous fishes and is conserved in the rest of jawed vertebrate clades. PAI-2 appears for the first time in the common ancestor of reptiles and mammals, and represents the latest appearing plasminogen activator inhibitor. Finally, we noted that the urokinase-type plasminogen activator receptor (uPAR)-and three-LU domain containing genes in general-occurred later in evolution and was first detectable after coelacanths.
This study identifies several primitive orthologues of the mammalian plasminogen activation system. These ancestral forms provide clues to the origin and diversification of this enzyme system. Further, the discovery of several members-hitherto unknown in mammals-provide new perspectives on the evolution of this important enzyme system.
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