The Application and Usefulness of Economic Analyses for Water Quality Management in Coastal Areas

Economic studies are increasingly sought as tools to contribute to water quality management in estuaries and coastal communities, yet little is known about how the results from existing studies have been received and utilized by the organizations who solicited them. We interviewed managers from eight organizations who solicited economic studies over the past 15 years to understand how useful the studies were to their organizations and what economic research would be most helpful for their management needs. In terms of utility for coastal managers, there are a number of limitations in the studies. These include lack of site-specific data, the high cost of thorough studies, the appropriate application of methods, and receiving highly technical information that can be difficult to translate to the appropriate stakeholder audiences. Despite these drawbacks, we found the managers to be extremely positive about the usefulness of the economic studies, but in need of more research and localized data. Managers who embark upon economic analyses should take care to engage trained economists who can identify and implement appropriate methodologies to answer management questions, and who can help managers to interpret and communicate the findings. The coastal managers also identified specific areas of research that are most salient for their programs. These range from broad applications of economic analysis as a communication tool, to specific applications such as cost-effectiveness analyses of management actions. Overall, the interviews revealed great interest and utility in economic analyses, and also opportunities for conducting specific economic analyses to improve coastal decision making

Unexpected activity of a novel kunitz-type inhibitor Inhibition of cysteine proteases but not serine proteases

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki ô 0.4-27 nM). FhKT1 prevented the autocatalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu15 within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu15 / Arg15 ) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki ô 1.5 nM). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu15 in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host

Fasciola hepatica is refractory to complement killing by preventing attachment of mannose binding lectin (MBL) and inhibiting MBL-associated serine proteases (MASPs) with serpins

The complement system is a first-line innate host immune defence against invading pathogens. It is activated via three pathways, termed Classical, Lectin and Alternative, which are mediated by antibodies, carbohydrate arrays or microbial liposaccharides, respectively. The three complement pathways converge in the formation of C3-convertase followed by the assembly of a lethal pore-like structure, the membrane attack complex (MAC), on the pathogen surface. We found that the infectious stage of the helminth parasite Fasciola hepatica, the newly excysted juvenile (NEJ), is resistant to the damaging effects of complement. Despite being coated with mannosylated proteins, the main initiator of the Lectin pathway, the mannose binding lectin (MBL), does not bind to the surface of live NEJ. In addition, we found that recombinantly expressed serine protease inhibitors secreted by NEJ (rFhSrp1 and rFhSrp2) selectively prevent activation of the complement via the Lectin pathway. Our experiments demonstrate that rFhSrp1 and rFhSrp2 inhibit native and recombinant MBL-associated serine proteases (MASPs), impairing the primary step that mediates C3b and C4b deposition on the NEJ surface. Indeed, immunofluorescence studies show that MBL, C3b, C4b or MAC are not deposited on the surface of NEJ incubated in normal human serum. Taken together, our findings uncover new means by which a helminth parasite prevents the activation of the Lectin complement pathway to become refractory to killing via this host response, in spite of presenting an assortment of glycans on their surface.</jats:p

Surface molecules of extracellular vesicles secreted by the helminth pathogen Fasciola hepatica direct their internalisation by host cells.

<div><p>Helminth parasites secrete extracellular vesicles (EVs) that can be internalised by host immune cells resulting in modulation of host immunity. While the molecular cargo of EVs have been characterised in many parasites, little is known about the surface-exposed molecules that participate in ligand-receptor interactions with the host cell surface to initiate vesicle docking and subsequent internalisation. Using a membrane-impermeable biotin reagent to capture proteins displayed on the outer membrane surface of two EV sub-populations (termed 15k and 120k EVs) released by adult <i>F</i>. <i>hepatica</i>, we describe 380 surface proteins including an array of virulence factors, membrane transport proteins and molecules involved in EV biogenesis/trafficking. Proteomics and immunohistochemical analysis show that the 120k EVs have an endosomal origin and may be released from the parasite via the protonephridial (excretory) system whilst the larger 15k EVs are released from the gastrodermal epithelial cells that line the fluke gut. A parallel lectin microarray strategy was used to profile the topology of major surface oligosaccharides of intact fluorogenically-labelled EVs as they would be displayed to the host. Lectin profiles corresponding to glycoconjugates exposed on the surface of the 15 K and 120K EV sub-populations are practically identical but are distinct from those of the parasite surface tegument, although all are predominated by high mannose sugars. We found that while the <i>F</i>. <i>hepatica</i> EVs were resistant to <i>exo</i>- and <i>endo</i>-glycosidases, the glyco-amidase PNGase F drastically remodelled the surface oligosaccharides and blocked the uptake of EVs by host macrophages. In contrast, pre-treatment with antibodies obtained from infected hosts, or purified antibodies raised against the extracellular domains of specific EV surface proteins (DM9-containing protein, CD63 receptor and myoferlin), significantly enhanced their cellular internalisation. This work highlights the diversity of EV biogenesis and trafficking pathways used by <i>F</i>. <i>hepatica</i> and sheds light on the molecular interaction between parasite EVs and host cells.</p></div

Research on Pancreas Disease in Irish Farmed Salmon 2004/2005 – Current and Future Initiatives

Pancreas Disease is the most significant single infectious disease affecting marine salmon farms in Ireland. The first epidemiological studies of Pancreas Disease (PD) in Ireland in the early 1990’s indicated that significant losses occurred in farmed Atlantic salmon in their first year at sea on some individual farms (Menzies et al., 1996). A serological survey for the presence of Salmon Pancreas Disease Virus (SPDV) antibody in 1996 revealed that 53% of Irish sites were positive, but that not all positive sites had recognised clinical signs of PD. This indicated a relatively low incidence and severity of PD at that time (McLoughlin et al., 1998) and this pattern persisted throughout the remainder of the 1990’s. In 2002, a serious increase in both the incidence and severity of PD was reported on farmed Atlantic salmon marine sites in Ireland. An epidemiological survey of Irish sites for 2002 revealed that 59% of sites reported an outbreak of PD, with mortalities averaging 12% (range 1 – 42%; McLoughlin et al., 2003). A recent study using data collected for 2003 and 2004 indicated that PD occurred in 62% and 86% of sites respectively. The average mortality due to PD on affected farms was 18.8% in 2003 and 14.8% in 2004. In response to these significant losses due to PD a range of research initiatives was launched in Ireland and was supported by the Marine Institute. This document gives an overview of the most recent findings from the current projects, carried out in 2004/2005 and also provides an overview of the various actions in Scotland and Norway, where PD has also become a disease of economic significance in salmon aquaculture.Funder: Marine Institut

Production of a functionally active recombinant SARS-CoV-2 (COVID-19) 3C-Like protease and a soluble inactive 3C-like protease-RBD chimeric in a prokaryotic expression system

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) intracellular life-cycle, two large polyproteins, pp1a and pp1ab, are produced. Processing of these by viral cysteine proteases, the papain-like protease (PLpro) and the chymotrypsin-like 3C-like protease (3CL-pro) release non-structural proteins necessary for the establishment of the viral replication and transcription complex (RTC), crucial for viral replication. Hence, these proteases are considered prime targets against which anti-coronavirus disease 2019 (COVID-19) drugs could be developed. Here, we describe the expression of a highly soluble and functionally active recombinant 3CL-pro using Escherichia coli BL21 cells. We show that the enzyme functions in a dimeric form and exhibits an unexpected inhibitory profile because its activity is potently blocked by serine rather than cysteine protease inhibitors. In addition, we assessed the ability of our 3CL-pro to function as a carrier for the receptor binding domain (RBD) of the Spike protein. The co-expressed chimeric protein, 3CLpro-RBD, did not exhibit 3CL-pro activity, but its enhanced solubility made purification easier and improved RBD antigenicity when tested against serum from vaccinated individuals in ELISAs. Chimeric proteins containing the 3CL-pro could represent an innovative approach to developing new COVID-19 vaccines

Diagnosis of sheep fasciolosis caused by Fasciola hepatica using cathepsin L enzyme-linked immunosorbent assays (ELISA)

Publication history: Accepted - 3 July 2021; Published online - 6 July 2021.Fasciolosis, a global parasitic disease of agricultural livestock, is caused by the liver fluke Fasciola hepatica. Management and strategic control of fasciolosis on farms depends on early assessment of the extent of disease so that control measures can be implemented quickly. Traditionally, this has relied on the detection of eggs in the faeces of animals, a laborious method that lacks sensitivity, especially for sub-clinical infections, and identifies chronic infections only. Enzyme linked immunosorbent assays (ELISA) offer a quicker and more sensitive serological means of diagnosis that could detect early acute infection before significant liver damage occurs. The performance of three functionally-active recombinant forms of the major F. hepatica secreted cathepsins L, rFhCL1, rFhCL2, rFhCL3, and a cathepsin B, rFhCB3, were evaluated as antigens in an indirect ELISA to serologically diagnose liver fluke infection in experimentally and naturally infected sheep. rFhCL1 and rFhCL3 were the most effective of the four antigens detecting fasciolosis in sheep as early as three weeks after experimental infection, at least five weeks earlier than both coproantigen and faecal egg tests. In addition, the rFhCL1 and rFhCL3 ELISAs had a very low detection limit for liver fluke in lambs exposed to natural infection on pastures and thus could play a major role in the surveillance of farms and a ‘test and treat’ approach to disease management. Finally, antibodies to all three cathepsin L proteases remain high throughout chronic infection but decline rapidly after drug treatment with the flukicide, triclabendazole, implying that the test may be adapted to trace the effectiveness of drug treatment.This work was supported by a European Research Council Advanced Grant (HELIVAC, 322725) and Science Foundation Ireland (SFI) Professorship grant (17/RP/5368) awarded to J.P. Dalton

Beyond the Mammalian Heart: Fish and Amphibians as a Model for Cardiac Repair and Regeneration

The epidemic of heart disease, the leading cause of death worldwide, is made worse by the fact that the adult mammalian heart is especially poor at repair. Damage to the mammal heart—such as that caused by myocardial infarction—leads to scarring, resulting in cardiac dysfunction and heart failure. In contrast, the hearts of fish and urodele amphibians are capable of complete regeneration of cardiac tissue from multiple types of damage, with full restoration of functionality. In the last decades, research has revealed a wealth of information on how these animals are able to perform this remarkable feat, and non-mammalian models of heart repair have become a burgeoning new source of data on the morphological, cellular, and molecular processes necessary to heal cardiac damage. In this review we present the major findings from recent research on the underlying mechanisms of fish and amphibian heart regeneration. We also discuss the tools and techniques that have been developed to answer these important questions