Environmental DNA as a tool to reconstruct catch composition for longline fisheries vessels.

Global wild-capture fisheries are a large and diverse sector requiring various tools for fisheries-dependant data collection and effective Monitoring, Control and Surveillance (MCS). Here we present a novel protocol to collect eDNA from brine tanks onboard commercial longline vessels to reconstruct catch composition. We collected samples from nine vessels operating out of the Eastern Tuna Billfish Fishery, Australia, validating eDNA results with reliable catch data consisting of seven target and bycatch species. Environmental DNA was highly effective for detecting species retained on vessels without contamination or false positives. For four vessels, logbook data and eDNA were consistent with detections of all species. The remaining vessels detected all species except for rare catches of short-billed spearfish (Tetrapturus angustirostris). Similarities between rank abundance distributions of catch and eDNA reads were observed with logbook data mirrored when eDNA sequences were organised into rank order abundance. The method was effective at identifying highly abundant taxa retained in brine tanks- tuna (Thunnus spp.), swordfish (Xiphias gladius), marlin (Kajijia audax), and Atlantic Pomfret (Brama brama). Further research is required to validate how eDNA and other molecular monitoring tools can be scaled and applied to provide solutions for monitoring challenges in the fisheries sector.

The hotspots of entanglement for pinnipeds of the world.

Pinnipeds represent one of the most vulnerable marine groups severely affected by entanglements. However, the lack of standardized data collection poses a challenge when comparing the impacts of fishing gear across various geographic regions. In this study, we employed Generalized Additive Models to predict entanglement incidents stemming from fishing-related activities for 13 pinniped species across the last four decades (1976-2017). The models incorporated reported entanglement numbers, fishing effort covariates based on different gear types, and floating plastic debris distribution for each species. Through this approach, we generated global hotspot maps that pinpoint regions of heightened vulnerability where pinnipeds are susceptible to entanglement in lost gear. The best-performing model highlighted both species characteristics and the presence of floating plastic debris as pivotal factors in predicting pinniped entanglements. Our analysis revealed entanglement hotspots in the North Pacific and Southeastern Australia. This demonstrates the efficacy of our methodology in identifying high-priority geographic areas.

State and local pressures drive plastic pollution compliance strategies.

Environmental harm from plastic pollution partly results from compliance failure at the individual level. Three prevalent non-compliant motivations for polluting plastics include economic gains, ignorance of the rules and unlikely penalization from inadequately enforced rules. Given compliance is primarily the responsibility of local waste management, we conducted interviews to gain insights to the factors driving changes in the crucial on-ground controls of plastic pollution. We expand on non-compliant motivations and provide a theoretical framework to test the aforementioned. We show that compliance strategies are strongly driven by state judicial and economic controls, specifically new plastic legislation and levies. Furthermore, the priorities of waste managers and the socio-economics and population density of their constituents drove changes in local management efforts. Our findings support the view that the growing global attention on plastic pollution shapes not only what happens at a state level, but also importantly on-ground at the local level.

Tools and constraints in monitoring interactions between marine litter and megafauna: Insights from case studies around the world.

Adverse impacts of marine litter is documented on >1400 species, including marine megafauna (fish, birds, sea turtles and mammals). The primary impacts include ingestion and entanglement, and there is increasing concern about chemical contamination via ingestion. Numerous survey approaches and monitoring programs have been developed and implemented around the world. They may aim to provide data about parameters such as species distribution and interactions with anthropogenic activities. During the Sixth International Marine Debris Conference, a session was dedicated to the tools and constraints in monitoring interactions between litter and megafauna. In the present paper, we summarize 7 case studies which discuss entanglement and ingestion including macro- and micro-debris in several taxa and across multiple geographic regions. We then discusses the importance of tools and standardizing methods for assessment and management purposes, in the context of international environmental policies and marine litter strategies.

Recombinant porcine sequence factor VIII (rpFVIII) for acquired haemophilia A: practical clinical experience of its use in seven patients.

INTRODUCTION: A recombinant porcine factor VIII B-domain-deleted product (rpFVIII; OBIZUR, Baxalta Incorporated, Deerfield, IL 60015, USA) was recently approved for treatment of bleeding episodes in adults with acquired haemophilia A (AHA) in the United States. To date, no clinical experience outside the registration study has been reported. AIM: To describe early clinical experience using rpFVIII for AHA. METHODS: A retrospective chart review of seven patients with AHA treated with rpFVIII at four institutions from November 2014 to October 2015. RESULTS: The time to diagnosis of AHA ranged from 5 days to 6 weeks. Six major and one other bleed were treated with rpFVIII following unsatisfactory bypassing agent (BPA) therapy. Good haemostatic efficacy was seen in five of seven cases. rpFVIII loading doses of 100 (n = 6) or 200 U kg-1 (n = 1) increased FVIII activity from <1 to 9% at baseline to 109-650% within 0.25-7 h in six of seven cases. Subsequent median doses ranged from 30 to 100 U kg-1 for 3-26 days. No rpFVIII-related adverse events were reported. Three patients survived with inhibitor eradication, one with persistent inhibitor, two died with inhibitors present and one was discharged and later died from unrelated causes. CONCLUSIONS: rpFVIII showed good haemostatic efficacy with no recurrences in most cases, with consumption substantially less than in the registration study. Treatment decisions were based on FVIII activity levels and clinical assessment. The ability to titrate rpFVIII dose using FVIII activity was considered advantageous compared with BPA therapy. Notable delays in diagnosis were observed.

Mistaken identity? Visual similarities of marine debris to natural prey items of sea turtles.

BACKGROUND: There are two predominant hypotheses as to why animals ingest plastic: 1) they are opportunistic feeders, eating plastic when they encounter it, and 2) they eat plastic because it resembles prey items. To assess which hypothesis is most likely, we created a model sea turtle visual system and used it to analyse debris samples from beach surveys and from necropsied turtles. We investigated colour, contrast, and luminance of the debris items as they would appear to the turtle. We also incorporated measures of texture and translucency to determine which of the two hypotheses is more plausible as a driver of selectivity in green sea turtles. RESULTS: Turtles preferred more flexible and translucent items to what was available in the environment, lending support to the hypothesis that they prefer debris that resembles prey, particularly jellyfish. They also ate fewer blue items, suggesting that such items may be less conspicuous against the background of open water where they forage. CONCLUSIONS: Using visual modelling we determined the characteristics that drive ingestion of marine debris by sea turtles, from the point of view of the turtles themselves. This technique can be utilized to determine debris preferences of other visual predators, and help to more effectively focus management or remediation actions.

A Value Chain Analysis of ghost nets in the Arafura Sea: identifying trans-boundary stakeholders, intervention points and livelihood trade-offs.

Lost or discarded fishing nets are a significant component of marine debris which has trans-boundary impacts in large marine ecosystems. Such 'ghost nets' cause the by-catch of marine fauna and require retrieval from coastlines where they wash up. Identifying the causes of discarded nets and feasible intervention points requires analysis of a complex value chain and the stakeholders within it, yet no studies have attempted this. In this paper we combine Value Chain Analysis, commonly applied to understand value-adding for a commodity, with elements of Life Cycle Assessment and social network analysis to examine the drivers, stakeholders, economic, environmental and social costs and benefits in the life of a trawl net. We use the Arafura Sea as a case study, which is shared by Indonesia, Papua New Guinea and Australia, and is the focus of a Trans-boundary Diagnostic Assessment (TDA) within the Arafura-Timor Seas Ecosystem Action program (ATSEA). We follow a trawl net through four sub-systems: manufacture of webbing in South Korea, fishing and loss by an Indonesian vessel, retrieval as ghost net on the northern Australian coastline by Indigenous rangers, and disposal or re-cycling as 'GhostNet Art' by Indigenous artists. Primary stakeholders along the value chain incur economic and social benefits, and economic and environmental costs. There is an anomaly in the chain between Indonesian fishermen and Indigenous rangers, artists and communities due to the lack of market linkages between these primary stakeholders. The first 'nexus of influence' where reductions in net losses and environmental costs can be achieved is through interactions between GhostNets Australia, the World Wide Fund for Nature and the Australian Government, which can influence Indonesian fishery management institutions and fishing crews. The second nexus is via the international art market which by publicising GhostNet Art can raise awareness amongst fish consumers about the impacts of ghost nets, and hence influence Indonesian fishing companies. GhostNets Australia is a key bridging organisation in the network, linking stakeholders across scales and sub-systems. Feasible preventative interventions are discussed to rectify the anomaly in the value chain. The importance of GhostNets Australia and ATSEA in the evolving adaptive co-management and trans-boundary governance of fisheries is highlighted. However, the prevention of ghost nets will result in trade-offs in benefits for the livelihoods of primary stakeholders. The utility of the method for analysing marine debris in TDAs, and ATSEA in particular, is discussed.

Variability in bleeding phenotype in Amish carriers of haemophilia B with the 31008 C-->T mutation.

The aim of this study was to characterize the variability of bleeding phenotype and its association with plasma factor IX coagulant activity (FIX:C) in haemophilia B carriers in a large Amish pedigree with a unifying genetic mutation, C-to-T transition at base 31008 of the factor IX gene (Xq27.1-27.2). A cross-sectional survey of haemophilia B carriers included a multiple choice questionnaire evaluating symptoms of mucocutaneous bleeding, joint bleeding and bleeding after haemostatic stress [menstruation, postpartum haemorrhage (PPH), dental extractions and invasive surgeries]. Severity of bleeding was graded as 0 to 4, 0 being no bleeding whereas 4 being severe bleeding. Association between total bleeding scores and the FIX:C was evaluated. Sixty-four haemophilia B carriers participated in this study. Median age: 18 years (range 1-70 years); median bleeding score: 1 (range 0-8). Besides PPH, isolated symptoms of bruising, epistaxis, menorrhagia and postsurgical bleeding including dental extraction were not associated with lower FIX:C. Bleeding score >/=3 was associated with involvement of at least two bleeding sites and a lower mean FIX:C of 42 +/- 10.3% (95% CI 36.4-47.7) while a score >3 had involvement of /=3. Phenotypic variability existed among the carriers of haemophilia B who belonged to a single pedigree carrying a single unifying mutation. The utility of bleeding scores to define bleeding phenotype precisely in haemophilia B carriers needs further evaluation.

Permanent Genetic Resources added to Molecular Ecology Resources database 1 January 2009-30 April 2009.

This article documents the addition of 283 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Agalinis acuta; Ambrosia artemisiifolia; Berula erecta; Casuarius casuarius; Cercospora zeae-maydis; Chorthippus parallelus; Conyza canadensis; Cotesia sesamiae; Epinephelus acanthistius; Ficedula hypoleuca; Grindelia hirsutula; Guadua angustifolia; Leucadendron rubrum; Maritrema novaezealandensis; Meretrix meretrix; Nilaparvata lugens; Oxyeleotris marmoratus; Phoxinus neogaeus; Pristomyrmex punctatus; Pseudobagrus brevicorpus; Seiridium cardinale; Stenopsyche marmorata; Tetranychus evansi and Xerus inauris. These loci were cross-tested on the following species: Agalinis decemloba; Agalinis tenella; Agalinis obtusifolia; Agalinis setacea; Agalinis skinneriana; Cercospora zeina; Cercospora kikuchii; Cercospora sorghi; Mycosphaerella graminicola; Setosphaeria turcica; Magnaporthe oryzae; Cotesia flavipes; Cotesia marginiventris; Grindelia Xpaludosa; Grindelia chiloensis; Grindelia fastigiata; Grindelia lanceolata; Grindelia squarrosa; Leucadendron coniferum; Leucadendron salicifolium; Leucadendron tinctum; Leucadendron meridianum; Laodelphax striatellus; Sogatella furcifera; Phoxinus eos; Phoxinus rigidus; Phoxinus brevispinosus; Phoxinus bicolor; Tetranychus urticae; Tetranychus turkestani; Tetranychus ludeni; Tetranychus neocaledonicus; Tetranychus amicus; Amphitetranychus viennensis; Eotetranychus rubiphilus; Eotetranychus tiliarium; Oligonychus perseae; Panonychus citri; Bryobia rubrioculus; Schizonobia bundi; Petrobia harti; Xerus princeps; Spermophilus tridecemlineatus and Sciurus carolinensis.

Characterization of microsatellite loci for the endangered cactus Echinocactus grusonii, and their cross-species utilization.

Echinocactus grusonii is common in trade but critically endangered in its natural habitat. With the ultimate aim of developing a certification scheme to aid in the conservation of this species, we have isolated E. grusonii microsatellites from a nonenriched library. Fifty-seven sequences contained a microsatellite array, of which 12 were polymorphic among 30 individuals from a single wild population. All 12 microsatellite primer pairs amplified product in one or more species in a screen of 27 other cactus species.

Characterization of microsatellite loci for the critically endangered cactus Ariocarpus bravoanus.

Ariocarpus bravoanus is common in trade but critically endangered in its natural habitat. With the ultimate aim of developing a certification scheme to aid in the conservation of this species, we have isolated A. bravoanus microsatellites from a nonenriched library. Fifty-four sequences contained a microsatellite array, of which eight were polymorphic among 23 individuals, 20 from one population and three plants from trade.

Spatial genetic structure of Simarouba amara Aubl. (Simaroubaceae), a dioecious, animal-dispersed Neotropical tree, on Barro Colorado Island, Panama.

Simarouba amara (Simaroubaceae) is a vertebrate-dispersed, insect-pollinated Neotropical tree found in lowland moist forest from upper Mesoamerica to the Amazon basin. We assessed the spatial genetic structure of S. amara within the 50-ha Forest Dynamics Plot on Barro Colorado Island in the Republic of Panama. A total of 300 individuals were genotyped using five microsatellite loci, representing 100 individuals with a dbh>or=10 cm, 100 individuals of 1-10 cm dbh, and 100 individuals of <1 cm dbh. The 200 individuals in the two larger size classes were also genotyped with 155 AFLP loci. Spatial autocorrelation analysis using Moran's Index detected significant genotypic association at the smallest distance classes for 1-10 cm dbh (0-20 m) and >10 cm dbh (0-40 m) size categories. Significant spatial autocorrelations were detected over larger scales (0-140 m) in <1 cm dbh individuals. The relatively weak genetic structure of S. amara, in comparison to other recent studies, may be explained by pollen and seed dispersal over the 50 ha plot, overlapping seed shadows, and postrecruitment mortality.

Cotranslational folding--omnia mea mecum porto?

Evidence for cotranslational folding on both prokaryotic and eukaryotic ribosomes is reviewed. Molecular chaperones appear to assist only a small fraction of newly synthesized proteins in folding into their native conformation. The recently published crystal structure of the large ribosomal subunit at 2.5 A resolution has provided the basis for understanding where and how peptide synthesis takes place on the ribosome. The nascent peptide is concluded to pass through a tunnel that extends about 100 A between the peptidyl transferase center and its exit site. The minimum diameter of the tunnel and the apparent physical and chemical properties of its walls appear to preclude complex folding of the nascent peptide within most of the length of the tunnel. However, results indicate that nascent peptides that are protected within the ribosomes vary in length from about 30 to 72 amino acid residues. This suggests that nascent peptides have different conformations. It is hypothesized that folding of the nascent polypeptide into its native conformation starts in the distal portion of the tunnel, and proceeds at the surface of the ribosomal subunit in a depression or bay near the exit opening of the tunnel.

An additional serine residue at the C terminus of rhodanese destabilizes the enzyme.

The rhodanese coding sequence was extended at its 3' end by three base pairs to generate mutants coding for a serine or arginine residue at the carboxyl terminus of the protein. Wild-type and mutant coding sequences were expressed in a cell-free Escherichia coli system by coupled transcription/translation. Predominantly full-length protein was formed in all cases. The amount of protein synthesized was quantified by incorporation of radioactive leucine into polypeptides. Enzymatic activity of in vitro synthesized rhodanese was determined at different temperatures. Specific enzymatic activity was calculated and is assumed to reflect the portion of the protein that is in its native three-dimensional conformation. It was observed that rhodanese extended by one serine at the C terminus lost enzymatic activity when incubated above 30 degrees C, in contrast to wild-type protein or variant rhodanese extended by an arginine residue. Similarly, variant rhodanese with an additional serine residue was more susceptible to urea denaturation than the other two rhodanese species. These results are surprising in light of the crystal structure of the protein.

Folding of a nascent peptide on the ribosome.

Even though very significant progress has been made recently in elucidating the structure of the bacterial ribosome and topological assignments of its functional parts, the molecular mechanism of how a peptide is formed and how the nascent peptides is folded on the ribosomes remains uncertain. Here, the current progress and remaining problems are considered from the standpoint of the authors. Topics considered include formation of peptide bonds and models that represent this process, the vicinity of RNA to the nascent peptide, the cotranslational folding hypothesis, evidence that some but not all nascent peptides pass through a region within the 50S ribosomal subunit, presumably the tunnel, in which they are folded and sheltered, pause-site peptides, and the involvement of chaperones in folding of nascent proteins on ribosomes. The chaperone-like activity of the large ribosomal subunit in renaturation of denatured proteins is reviewed. It is concluded that cotranslational folding of some but not all nascent peptides occurs in the large ribosomal subunit. It is suggested that this folding is facilitated by changes in the conformation of the ribosome that are related to the reaction cycle of peptide elongation.

Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes.

Five different coding sequences of bacterial or eukaryotic origin in plasmids under the T7 promoter were expressed in a cell-free system derived from Escherichia coli. Translation on E. coli ribosomes resulted in a full-length product only in four of the five coding sequences tested. A unique pattern of less than full-length polypeptides was generated in each case. Many of these polypeptides on E. coli ribosomes reacted with a puromycin derivative, cytidylic acid-puromycin, which was radioactively labeled. Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site. Certain nascent peptides could be shifted into full-length protein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell-free system resulted in a 80-90% full-length product with no evidence for nascent polypeptides and translational pausing.

Initiation of protein synthesis with fluorophore-Met-tRNA(f) and the involvement of IF-2.

The complicity of initiation factor 2 (IF-2) in causing the observed low incorporation of N-terminal fluorophore from fluorophore-methionyl-tRNA(f) during protein synthesis in an in vitro coupled transcription/translation system was investigated. The low incorporation in comparison to formyl-methionine was not due to the lack of interaction of fluorophore-Met-tRNA(f) with IF-2. Fluorescence measurements of cascade yellow-, eosin-, pyrene-, or coumarin-Met-tRNA(f) determined that all were capable of binding IF-2 at 4 mM Mg(2+) and 37 degrees C. Filter binding assays conducted in the absence of magnesium ions on fMet-tRNA(f), eosin-Met-tRNA(f), and cascade yellow-Met-tRNA(f) confirmed the previously reported value for the dissociation constant of fMet-tRNA(f) of about 1 microM and placed the binding constants for the two fluorophore derivatives about three-fold higher. Binding of the fluorophore-Met-tRNA(f) species to salt-washed ribosomes showed a more significant decrease compared to fMet-tRNA(f). Stimulation in the amount of tRNA bound to the ribosomes upon the addition of IF-2 was observed in each case. All ribosome-bound cascade yellow-Met-tRNA(f) and eosin-Met-tRNA(f) were as puromycin-reactive as fMet-tRNA(f). Cumulatively, the effects observed for the fluorophore-Met-tRNA species in partial reactions of initiation may account for the reduced incorporation of these probes at the N terminus of polypeptides.

Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome.

Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.

Domain separation precedes global unfolding of rhodanese.

The enzyme rhodanese was investigated for the conformational transition associated with its urea unfolding. When rhodanese was treated with 0 or 3 M urea, the activity was not significantly affected. 4.25 M urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6 M urea. The 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 M urea equilibrations, and the fluorescence was dramatically increased with 4.25 M urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 M urea equilibration, rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 M urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of rhodanese.

N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins.

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indicated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form may be inactivated without being degraded. These experiments were carried out by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-terminal and C-terminal modifications of the coding sequence on the ribosomal release/termination process and on the stability of the newly synthesized protein. Elimination of the leader sequence in two proteins tested, mitichondrial rhodanese and bacterial beta-lactamase, caused an increase in the percentage of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-lag impaired the ribosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribosomes. A C-terminal extension of the coding sequence for rhodanese by one amino acid decreased the percentage released polypeptide and furthermore affected the stability of the in vitro formed protein. We propose that a regulatory mechanism exists by which N-terminal and C-terminal sequences of a newly synthesized protein have feed-back effects on the termination factor-mediated release and on the stability of the native three-dimensional structure.